As a greater number of research teams begin to use molecular technology rather than standard cultivation mechanisms to detect bacteria in their samples, it is becoming increasingly obvious why doctors and researchers are unaware that their patients with chronic inflammatory disease are infected with large quantities of L-form and biofilm bacteria – the techniques they are using to look for bacteria prove rather useless in actually identifying the pathogens.

This month yet another paper was published whose results confirm that the Petri dish is a thing of the past. The study, which was published in BMC Microbiology, used a series of molecular techniques to identify the species of biofilm bacteria present inside several different kinds of wounds.[1]

Bioflims are formed when a complex and varied group of bacteria aggregate together inside a protective and adhesive protein matrix. The bacteria inside a biofilm cooperate to promote their own survival and the chronic nature of an infection. While dentists have long realized that bacteria in the mouth often reside inside biofilms – they form much of the goo that they remove from teeth – researchers are just starting to investigate bioflim communities that form in other areas of the body.

Since few studies have performed extensive surveys of the bacterial populations within different types of chronic wound biofilms, the BMC team, comprised of individuals from the United States Department of Agriculture, the Medical Biofilm Research Institute in Texas, and the Center for Biofilm Engineering at Montana State University, used several advanced molecular techniques to survey the major populations of bacteria in the pathogenic biofilms of three types of chronic wounds:

  • diabetic food ulcers
  • venous leg ulcers
  • pressure ulcers

The researchers used several different molecular methods – ribosomal amplification and cloning, Sanger sequencing (FRACS), partial ribosomal amplification, density gradient gel electrophoresis (DGGE), and Sanger sequencing (PRADS) – to provide a comprehensive survey of microbial populations.

Together, these molecular techniques revealed that some specific populations of bacteria were evident in the biofilms of all chronic wound types. These bacteria included Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia.

But there were also marked difference between the bacterial populations in each of the wounds. For example, in pressure ulcers, 62% of the populations identified were from a class of bacteria called obligate anaerobes – bacteria that do not require oxygen for growth and may even die in its presence.

In fact, every wound that the research team tested with new molecular technology was also tested for the presence of bacteria using traditional based culture methods. When the results of molecular analysis were compared to the bacteria obtained using these traditional methods, the team found that culture methods were only able to correctly identify the primary bacterial population in one wound type.

“Here we show that culturing failed to identify major contributing populations, especially strict anaerobes, within the given wound types,” state the team, “Standard culturing techniques are inherently biased as they only examine only the 1% of all microorganisms which are able to grow fairly rapidly in pure culture.”

The team also commented on the fact that it takes several days for bacteria grown by culturing methods to be identified, whereas molecular methods such as PCR can typically be completed within several hours. They also stress the fact that some forms of bacteria can simply not grow under the conditions required for standard cultivation, stating that “In addition, certain of the isolates that we have shown to be primary populations within a wound type may never be cultured in the laboratory due to reduced metabolic activity, obligate cooperation with other bacteria, requirements for specialized nutrients, or growth in specific environmental conditions.”

They also argue that molecular methods, unlike culture methods, have more potential to provide quantitative data, stating “arguably, we have shown that molecular methods will allow populations residing within biofilms to be more fully characterized.”

Furthermore, they make it clear that doctors will greatly benefit by knowing the exact composition of bacteria that each of their individual patients harbors. “The continued development of molecular methods may lead to vastly improved tools or diagnostics that will identify and provide quantification of the diverse species potentially present in chronic wounds thereby allowing physicians to better tailor their treatment to each patient’s unique pathogenic biofilm populations,” states the team.

Thus, much like other teams who are using molecular methods to detect bacteria, the researchers make an urgent call for the standardized use of improved diagnostic methods. Not only that, but they also hope the results of their study might actually “foster the pioneering, and development of new diagnostic tools.”

One of the study’s findings that also bears pertinence to the Marshall Protocol is the fact that the bacterial species Staphylococcus was found inside all chronic wound types. Two of the other largest studies to date to seek bacteria using molecular technology (one which looking at bacteria removed prosthetic joints, and another that looked at bacterial populations in patients with Cystic Fibrosis) also found Staphylococcus to be a common pathogen in their samples. Since minocycline is one of the antibiotics that best targets Staphpylococcus, these findings suggest that the decision to use minocycline as the base antibiotic for the Marshall Protocol was well warranted.

Also interesting is that researchers at the University of Turku in Finland just released the results of a study which found that infants who harbor theStaphylococcus aureus are more likely to become overweight and obese later in life. [2] Again, this suggests that Staphylococcus is a common cause of Th1 symptoms, and that obesity should respond to the MP antibiotic regimen that allows the bacterial species to be killed.


  1. Dowd, Scot et al. (2008). “Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing.” BMC microbiology 8(1):43. []
  2. Kalliomäki, Marko et al. 2008. “Early differences in fecal microbiota composition in children may predict overweight.” The American journal of clinical nutrition 87(3):534-8. []