Forty years ago, Dr. Alain Rambach invented chromogenic media, which may be the most widely used and impactful microbiological method for the identification of a large cross-section of invisible microbes, including those important to the medical world. Like many scientists, Rambach took an idea, a small seed of intuition, and nurtured it through various trials and tribulations until his dream was realized. His original chromogenic agar has been developed into an entire CHROMagar™ product line, and spawned an industry of competitors. Through a series of conversations with Rambach, we learned how his career in biological sciences evolved and how he developed his chromogenic agar, which has had a great impact on the field of clinical microbiologyover the last 25 years. 

From Mathematics to Genetic Engineering 

You might be surprised to learn that upon his graduation from École Polytechnique in France in 1965, Alain Rambach held a degree in mathematics, not biology. In fact, Rambach himself will tell you that he had little knowledge of biology, knowing only what he had read from textbooks by authors like Joël de Rosnay and James Watson. As fate would have it, Rambach joined the Institut Pasteur in 1967 and began working on a Ph.D. in bacterial genetics alongside Nobel Prize-winning scientist François Jacob.

Rambach’s work focused on the lambda bacteriophage of Escherichia coli, which would become a staple of his work for the next 50 years. After the defense of his thesis, Rambach recounts that in early 1973, he explained his future research goals to his mentorship team by drawing DNA on a chalkboard using 2 colors of chalk: blue chalk for the existing structure and orange chalk for the hypothetical insertion of new DNA. This vision of genetic manipulation, according to Rambach, was a novel and somewhat controversial concept at the time.

Meeting of the Minds

After spending a year (1973-1974) working on genetically modifying the lambda bacteriophage of E. coli to make it into a DNA cloning vector, Rambach completed a postdoctoral fellowship in biochemistry at Stanford University and returned to Institut Pasteur in 1978. He was appointed to the genetic engineering unit, where he focused on the medical diagnosis of bacterial infections, particularly infections caused by organisms belonging to the order Enterobacterales. 

It was in the genetic engineering unit that Rambach met Dr. Jean Buissiere, a military doctor who was completing 2 years of training in infectious disease diagnostics next door. Buissiere’s work focused on how chromophores, the part of a molecule that is responsible for the color we see, could be used to identify bacteria. In a short autobiography, Rambach recalls Buissiere teaching him the fundamental idea that by “characterizing the enzymatic equipment of bacteria, it was possible to obtain bacterial identification.” In 1976 for example, Kilian and Bulow found that the enzyme beta-glucuronidase is strongly associated with, and in fact confined to,  Escherichia coli. Since approximately 95% of E. coli strains demonstrate this enzymatic activity, detecting the presence or absence is indicative of whether E. coli itself is present. One way to easily test for beta-glucuronidase activity is to look for the production of a yellow chromophore hydrolyzed from a synthetic substrate. 

While most of this type of research was being performed in liquid broth tubes with pure cultures of bacteria, Rambach thought it would be advantageous to put the process into solid media. He hypothesized that if he used a chromophore such as indoxyl, which does not diffuse readily like the substances that were being used in broth culture at the time, it would be possible to identify various bacterial types by the color of their colonies, even when analyzing complex cultures from sources like clinical samples or food. The science behind the idea works like this:

1. The media contains molecules called chromogens. A chromogen molecule consists of a substrate (the ‘key’ to a specific enzyme ‘lock’), as well as a chromophore. The chromogen is colorless because the chromophore does not absorb visible light while conjugated to the substrate.
2. When a bacterial organism with specific enzymatic activity comes into contact with the chromogen molecule, that enzyme cleaves the chromogen molecule, releasing the chromophore.
3. When the chromophore is not conjugated, its color becomes visible. By using a chromophore that does not diffuse readily into the surrounding meida, the color stays concentrated in the area where the bacterial colony with the target enzymatic activity grew. Thus, the colony itself takes on the chromophore's color. 

With encouragement from Buissiere, Rambach decided to create this media himself. 

Chromogenic Media Is Born in 1979

Rambach started with the organism he had always worked with: E. coli. Knowing that glucuronidase is very specific to E. coli, Rambach mixed nutrient medium with a corresponding substrate called hydroxyquinoline-glucuronide, a substance that was obtained, at the time, by extracting it from the urine of dogs that had been given hydroxyquinoline. The process worked, and Rambach was able to demonstrate proof of concept. Despite his best efforts, no one was interested in this new technology at the time. The rejection was severely disappointing for Rambach, who said “my experience in bacterial diagnosis by improvement of a manual microbiology method end[ed] in failure.”

Diagnostics Research Meets Entrepreneurship

Manufacturers failed to see the potential in Rambach’s E. coli identification system (COLITEST™) in 1979. He had all but given up on developing diagnostic chromogenic media. After dedicating years to genetic engineering work, including founding an applied genetic engineering company in 1980, Rambach remained hopeful that his chromogenic media concept would take off and he continued to pursue industrial development. In 1989, he approached many large manufacturers, such as Becton Dickinson, Merck and Kodak, equipped with an arsenal of ideas, including plans for Rambach™ chromogenic Salmonella agar, as well as ambitious plans for additional chromogenic media.

Once again, Rambach met with disappointment. Not only were his plans rejected by all companies he approached, some even asked him to stop contacting them. Rather than succumb to defeat, Rambach decided to launch the Salmonella agar himself under the name of Rambach™ agar. The move was successful, and Rambach continued to develop and innovate in the years that followed:

• In 1992, Rambach developed additional media formulas for detection of E. coli that provided a variety of colors from blue-gray to red. 
• In 1994, Rambach developed the first CHROMagar™ bichromogenic medium. By combining 2 chromogens, this media differentiates several organisms at once using several colors. 
• Also in 1994, the CHROMagar™ Candida plate was developed in order to identify various yeast species. 
• The European subsidiary of the American company Becton Dickinson asked if they could buy the powder to make these products and sell the media under the name BBL™ CHROMagar™, which Rambach accepted. 
• In 1997, Becton Dickinson asked Rambach to sign a work license contract allowing them to manufacture the powder themselves. Rambach agreed, so long as the contract included his current inventions as well as future inventions, and that the product was marked with the CHROMagar™ logo in the spirit of co-promotion. This relationship has been working successfully for the last 25 years.  

CHROMagar™ in the Clinical Microbiology Laboratory

The magnificent diversity in bacterial life can make the clinical microbiology laboratory feel like a kitchen where the chef (microbiologist or medical laboratory professional) must understand the recipe and preferences for growth of many individual microbes. However, these very general preferences tend to be the same for many microbes, when the ultimate goal is to culture, isolate and identify one particular group of bacteria or even one individual species. Chromogenic media, with which we can quickly “screen for” (culture and differentiate) a particular genus or species of bacteria, speeds up and scales the identification process. By speeding up this process, the clinical microbiologist can aid not only in diagnosing which infection a patient may have, but also in the selection of the appropriate antibiotic therapy for eventual treatment and cure.

For example, Staphylococcus is a genus of gram-positive cocci with a number of species, including Staphylococcus aureus, a pathogen that can cause infections ranging from simple (pimples and boils) to life-threatening (sepsis and pneumonia), and the coagulase negative staphylococci (CoNS), which are considered normal skin flora and rarely cause dangerous infections. Traditionally, staphylococci respond well to treatment with a class of antibiotics known as beta lactamase-resistant penicillins, such as methicillin. Unfortunately, strains of S. aureus have developed resistance to this group of antibiotics, and are known as methicillin-resistant S. aureus (MRSA). 

It is imperative that the medical laboratory rapidly identify MRSA to determine the best treatment regimen and avoid antibiotics that will not work. However, traditional microbial culture and antibiotic susceptibility testing can be time-consuming and complex. Chromogenic media designed to identify MRSA, including CHROMagar™ MRSA that was introduced in 2002 with sensitivity and specificity values close to 100%, all use basic principles designed by Rambach. These media contain S. aureus-specific substrates that the bacteria hydrolyze, allowing S. aureus colonies to be easily visualized. They also inhibit the growth of methicillin-sensitive S. aureus, ensuring that only MRSA colonies grow on the plate. Due to its reduction of both workload and turnaround time, this type of chromogenic medium offers a very rapid and large-scale screening option for a pathogen whose timely identification is crucial for patient care.

Similarly, rapid identification of Salmonella is not only important for the patient, but is crucial for public health surveillance and rapid containment of outbreaks, particularly from food sources. The Centers for Disease Control (CDC) estimates that Salmonella causes 1.35 million infections, 26,500 hospitalizations and 420 deaths in the United States each year.  

There are a variety of chromogenic agars available, including CHROMagar™ Salmonella Plus, for detecting Salmonella, and all demonstrate impressive sensitivity and lead to a reduction in workload and costs. These media are easy to interpret, even in the context of large numbers of E. coli bacteria (often found in very high numbers with Salmonella). Importantly, CHROMagar™ Salmonella Plus includes detection of uncommon lactose-positive Salmonella, which would not be selected for further study if only traditional, lactose-based media types were used (MacConkey and Hektoen agars, for example). The identification of lactose-positive Salmonella is required in food microbiology by the ISO 6579-1 standard.

In the United States, Candida yeast are some of the most common organisms causing hospital-associated bloodstream infections. While C. albicans is the most frequently isolated culprit, non-albicans species have become more common in recent years and are associated with higher mortality and higher rates of resistance to antifungals compared to C. albicans. Due to the slower rate of growth of yeast species, antimicrobial susceptibility testing can take considerably longer than for bacteria. Knowing which type of Candida species is growing in culture can help a clinician select empiric antifungal treatment, which is imperative in cases of serious infection, such as fungemia. 

Chromogenic media for the detection and differentiation of Candida species, such as CHROMagar™ Candida, are important tools for identifying major clinically-significant Candida species. Importantly, these media display powerful sensitivity and specificity for 3 of the most clinically significant species — C. albicans, C. tropicalis and C. krusei — of 99 % or higher. On these media, Candida albicans appears green, Candida tropicalis appears metallic blue, Candida krusei appears pink and fuzzy and other species appear white to mauve.

The Future of Chromogenic Agar

Rambach continues to play a central role in the management, research and development of CHROMagar™ products. The company boasts an ongoing commitment to quality science, with over 50% of its employees involved in research. The current mission of the organization is to find rapid detection and identification solutions for major infection control challenges, such as multidrug-resistant bacteria. If anything, Rambach’s story teaches us that novel ideas in science are not always popular. He is an example of how having enough determination and passion for a subject, plus perseverance, just may help you change the world. 

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Submit a Masterpiece to ASM Agar Art 2020! 

Chromogenic agar has been a popular canvas for agar art over the years, with several amazing pieces each year submitted to ASM's Agar Art Contest. If the beautiful colors of chromogenic agar left you inspired, check out this year's contest (we're allowing participants to use a broad range of artistic, not just microbial, media to create their artwork this year) and submit your work of art by Oct. 23, 2020.