“Bacterial L-forms are among the most unusual creatures in nature. Once one has seen their strange habits and life style, one starts to work on L-forms with great enthusiasm because their existence in vivo and in vitro gives rise to more questions in classical microbiology, immunology and infectious diseases.

Researching L-forms is like trying to catch a fish that appears on the surface and quickly dives back into the sea. It is almost impossible to catch it with bare hands. There are better chances of catching the fish by hook and line but chance is always powerful when efforts are joined and fishing nets are put into use.”

And just who is able to describe the process of culturing L-form bacteria so eloquently? She’s an Associate Professor at the Department of Pathogenic Bacteria Institute of Microbiology at the Bulgarian Academy of Sciences, who’s worked with L-form bacteria for the last 15 years. Meet Nadya Markova.

1. What led you to become interested in L-form bacteria?

Nadya Markova

I graduated as a medical doctor, but my interest in microbiology led me to the Bulgarian Academy of Sciences (where I defended my PhD thesis in the field of medical microbiology). Researchers already working there at the time had great experience in L-form research and had made many interesting observations about the bacteria, all of which sparked my interest. They were my teachers, who inspired me to continue their research in the same field.

2. How long have you been working with L-form bacteria? How many people do you currently work with and how to they contribute to the research environment?

I started thinking about how L-form bacteria change form in the beginning of the 90s, and my interest in them has risen ever since. Unfortunately, our research team is comprised of only 5 people. One of them must be mentioned, my teacher Professor Lilia Michailova. She is an excellent electron microscopist and, without her, our achievements wouldn’t have been possible. I’m really glad that she is still active and that we continue to work together.

3. Is your team working (or in the past has focused on) any particular species of L-form bacteria?

L-form bacteria as seen under an electron microscope

L-form bacteria as seen under an electron microscope; this photo and others taken by Nadya Markova

We have conducted various experiments with L-forms of Staphylococcus aureus, Streptococcus pyogenes, and Listeria monocytogenes. At present we are very interested in, and work exclusively with, L-forms ofMycobacterium tuberculosis.

4. How do you grow the L-form bacteria that you observe in the lab? What techniques do you use? What type of microscopes do you use to observe them?

We grow L-forms on specific media (semisolid and liquid) with supplements specifically designed to facilitate L-form growth. These media are well known in the literature and are widely used by other researchers. We observe L-forms using electron and light microscopes. However, in order to correctly observe L-form bacteria using a light microscope, a person must have a great deal of experience with L-forms.

5. What aspects of L-form bacteria has your research focused on?

We focus on bacterial host cell interactions, especially interactions of bacterial L-forms with peritoneal and alveolar macrophages in experimental infections.

6. Could you explain that to me in simple terms?

Samples showing L-forms multiplying in number

Samples showing L-forms multiplying in number

There are two types of experimental infections. The first involves dividing experimental animals in two groups. We inject the first group of animals with normal pathogenic bacteria and the other group with L-form strains. We grow the L-forms under specific laboratory conditions prior to the experiment.

During the experimental infection, we collect samples of white blood cells called alveolar macrophages in the animal’s lungs, and white blood cells called peritoneal macrophages from the abdomen of each animal. Macrophages are cells of the immune system that are supposed to be the first to react to infections. Their job is to engulf and digest the pathogens and then, in turn, create an antigen, a special type of molecule that provokes a response from the host’s immune system.

Phagocytosis is the process of engulfing, digesting, and transforming pathogenic bacteria into antigens. In the case of normal pathogenic bacteria, macrophages easily recognize the bacteria and the process of phagocytosis goes smoothly. But when macrophages are faced with L-form bacteria, they have difficulty recognizing the bacteria as pathogenic, and the normal process of phagocytosis is impeded.

L-forms stay longer on the surface of macrophages without being engulfed. Even when they are engulfed, L-forms are not properly digested and they continue to persist inside the macrophage. In short, the whole process of destroying bacteria is distorted.

It is important to understand that these phenomena happen in vivo, or inside animals that are alive.

7. What is one of the hardest parts about culturing L-form bacteria?

Nadya Markova and colleague, Professor Lilia Michailova

Nadya Markova and colleague, Professor Lilia Michailova

The hardest part is not so much cultivating the bacteria, but rather developing the skills to recognize which bacteria are L-forms. The importance of a teacher passing down this knowledge to a student is critical – this cannot be learned from books or photographs; one has to see it in person and have it explained.

8. What role to you think L-form bacteria play in causing certain chronic diseases? What, if any, diseases do you think they might cause?

In my opinion, long-lasting persistent irritation of the immune system by these unusual bacterial L-forms is the cause of most chronic diseases. I wouldn’t speculate and mention specific diseases, but I support the opinion that L-forms could be the reason for latent, chronic, and relapsing infections, as well as diseases of unknown infectious-allergic or autoimmune origin.

9. What do you feel are some of the most fascinating aspects of L-form bacteria?

L-form bacteria are so unusual, so it is hard for me to decide what I feel is most fascinating about them. I’m always amazed at how they can drastically change their life style. By life style, I mean that each microbe has its own shape and size, or its own morphology. Microbes are mainly shaped like rods or, shaped like spheres (called cocci). Usually microbes multiply by simple division, that is, each microbe divides into two new microbes which are morphologically identical.

But when it comes to L-forms, the process of division does not occur in the same, usual way. L-forms stop producing cell walls and start creating morphological units such as large bodies, elementary bodies, filaments, filterable granules, empty bodies, vesicles and membranous structures. When these diverse populations of L-forms get into an organism, they do not behave like other populations of normal pathogenic bacteria. They no longer interact in a normal manner with cells from the immune system.

Large bodies of the L-form Staphylococcus

Large bodies of the L-form Staphylococcus

Perhaps another one of the most important aspects about the pathology of L-form characteristics is that they are exceptionally resistant to many factors, including most antibacterial drugs and bactericidal host defense mechanisms. Simply speaking, the bactericidal host defense mechanism is the ability of macrophages to engulf and destroy pathogens and in the process create an antigen. It is the only way that the organism can produce a normal immune response to the bacterial threat.

10. What would you say has been your greatest or most interesting discovery about L-forms?

We are fascinated by, and focused on, observing and documenting the atypical behavior of L-forms in vivo, or in the body rather than the lab.

11. What do you feel are some of the most misunderstood aspects of L-form bacteria?

One of the main misconceptions about L-form bacteria is that they are considered to be an artificially provoked phenomenon capable of existing only in laboratory conditions. But a number of studies have shown that L-forms can exist in vivo (inside the body) as well.

12. Over the years, how has your work been received?

It’s hard to get our papers published, because few people actually understand and want to accept our investigations concerning bacterial L-form transformations. Unfortunately, this continues to this very day.

13. If you could briefly talk to the leader of the World Health Organization about L-form bacteria, what would you tell him?

Filamentous L-forms of M. tuberculosis

Filamentous L-forms of M. tuberculosis

I would tell him what an extremely important role L-form bacteria play in latent, chronic, and relapsing infections, as well as in diseases of unknown infectious-allergic or autoimmune origin. I would also tell him, if possible, to support and sponsor the researchers working with L-form bacteria, as they are mostly ignored.

14. In Bulgaria, what is the prevailing opinion about the cause of chronic disease?

Unfortunately, physicians in Bulgaria, and probably in other countries, don’t pay enough attention to the role of L-form bacteria in chronic diseases, and accept them with difficulty.

15. Are you in contact with other researchers who work with L-form bacteria?

The founder of L-form research in Bulgaria – Professor Toshkov – had great contacts and collaboration projects with L-form research teams in France, Germany and Russia, as well as contacts with famous scientists like Lida Mattman. Nowadays, there are relatively few people with whom I am in contact. However, no matter how hard it is, I’m doing my best to get in touch with other scientists working with modern molecular biological tools. I want to spark their enthusiasm so that they too will work in L-form research, which will allow this area of medicine to achieve real success.

16. If people don’t accept your research, what reasons do they give for saying you are incorrect? Does this frustrate you?

Spherical-shaped L-forms of M. tuberculosis

Spherical-shaped L-forms of M. tuberculosis

When reviewers don’t accept our papers, one of the main reasons they give is that our results are not supported with standard molecular-microbiological methods, which is why they refuse to accept them as real. It is very hard to explain to them that those standard methods for classical bacteria are not valid for L-form cells, and that new methods and approaches need to be developed in order to identify them using DNA- based technology.

17. If you could change one aspect of the current medical climate,
what would it be?

I think that it is very important to establish better cooperation and contacts between practicing physicians and those microbiologists working in laboratories that specialize on the isolation, cultivation, and identification of L-forms.

18. Have you heard about the Marshall Protocol? If so, how do you feel about the treatment?

Yes, I have heard about the Marshall Protocol and I am aware of the basic principles of the treatment. I feel that the approach of the Marshall Protocol, which first acts on the immune system and then continues the healing process with an extended etiotropic low-dose antibiotic therapy, is correct. In my opinion, at this stage of our understanding of L-form behavior, minocycline is one of the best antibiotics that is best able to suppress the activity of L-forms. However, I feel that all researchers working in the field of L-forms should be concentrating all their energy on developing new drugs that can specifically target and block the process of L-form conversion.

19. Does your team have any leads or ideas on how to go about creating a medicine that would block L-form conversion?

In order to create an effective medicine we have to profoundly investigate and fully understand the mechanism of L-form conversion (how normal pathogenic bacteria turn into L-forms) on the molecular and genetic level. Then we will be able to determine the exact level at which we can act in order to stop the process.

PAPERS

Michailova L., Kussovski V., Radoucheva T., Jordanova M. & Markova N. (2007). Persistence of Staphylococcus aureus L-form during experimental lung infection in rats. FEMS Microbiol Lett 268: 88-97.

Michailova L., Kussovski, V., Radoucheva, T., Jordanova M., Berger W., Rinder H. & Markova N. (2005). Morphological variability and cell wall deficiency in Mycobacterium tuberculosis ‘heteroresistant’ stains. The Int. J. Tuberc. Lung. Dis. 9: 907-914.

Michailova, L, Markova N., Radoucheva T., Stoitsova S., Kussovski V. & Jordanova M.. (2000). Atypical behaviour and survival of Steptococcus pyogenes L-forms during intraperitoneal infection in rats. FEMS Immunology and Medical Microbiology 28: 55-65.

Michailova, L., S. Stoitsova, N.Markova, T. Radoucheva, V.Kussovski, M. Jordanova, & Dimova. I. (2000). Interaction of alveolar macrophages with Staphylococcus aureus and induction of microbial L-forms during infection in rats. Int. J. Med. Microbiol. 290:259-267.

Markova N., Mihailova L., Vesselinova A. , Kussovski V., Radoucheva T., Nikolova S., & Paskaleva I. (1997). Cell wall deficient forms (L-forms) of Listeria monocytogenes in experimentally infected rats. Zbl. Bakt. 286: 46-55.

Mihailova, L., Markova, T., Radoucheva, D., Veljanov, S., & Radoevska. (1993).Cell interaction of Listeria monocytogenes L-forms and peritoneal exudative cells in rats. Can. J. Microbiol. 39: 1014-1021.