Tale of Gram’s Serendipitous Discovery

While working with infected lung tissue from cadavers, a Danish pathologist stumbled upon a remarkable technique still commonly used over 100 years later. 

How did this serendipitous discovery come about?

One fine day, while investigating the lungs of deceased patients with pneumonia in 1884. In the first stage, he poured Gentian (crystal) violet solution over a fluid smear on a glass slide and dried it over a burner flame. Lugol’s potassium triiodide solution was used as a mordant after a thorough rinse with water. He then used ethanol to remove the dye from the slide. Though the alcohol decolorized most bacteria, some, like pneumococci, kept their original color (gram-negative). Streptococcus pneumoniae and Klebsiella pneumoniae were the subjects of his initial experiments with this staining method.

Gram noticed that the bacterial cell wall composition is responsible for the variations. Peptidoglycan is a sugar and amino acid polymer found in some bacteria’s cell walls. Under the microscope, these “gram-positive” bacteria appear purple or brown because they have retained the colour of a stain (often a combination of crystal violet and iodine or methylene blue). And then there are the red ones, which are gram-negative bacteria because they lack peptidoglycan and hence cannot be stained with the corresponding blue dye. 

What is Gram Staining and How Does it Work?

A culture and Gram stain are carried out if an infection is suspected of identifying bacteria. If bacteria are present, this test can determine whether they are gram-positive or gram-negative. The difference between gram-negative and gram-positive bacteria can influence the treatment plan.

Gram stains can be performed on the following types of specimens: 

If there are signs of an infection, a gram stain is performed. They might not know whether or not the infection is bacterial, viral, fungal, or parasitic. In general, these sorts of conditions are treated differently. Different forms of bacterial infections may also need various interventions.

A gram stain can determine whether bacteria are responsible for symptoms and identify the types of bacteria present. 

Gram-Negative Bacteria & its Diseases

A protective capsule encloses Gram-negative bacteria. This capsule prevents infection-fighting white blood cells from ingesting bacteria. Gram-negative bacteria have an outer membrane that protects them against antibiotics such as penicillin. This membrane, when compromised, releases toxins known as endotoxins. Endotoxins contribute to the severity of symptoms caused by gram-negative bacterial infections.

Gram-negative bacteria can cause severe human diseases, particularly those with impaired immune systems. Due to AMR, nosocomial infections caused by Gram-negative bacilli (GNB) pose the most significant challenge to healthcare professionals.

Gram-negative bacteria resistant to antibiotics, including Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii, Salmonella spp, Neisseria gonorrhoeae, Haemophilus influenza, Campylobacter, Helicobacter pylori, and Shigella spp, constitute a threat to public health and the economy. 

AMR: An International Priority and Challenge

Antimicrobial resistance is a massive worldwide health concern and one of the greatest threats to humanity today. Some strains of bacteria have developed resistance to nearly all antibiotics. Therefore, new antibacterial drugs are essential to counter resistant microorganisms. The World Health Organization (WHO) has put together a list of antibiotic-resistant organisms that pose a significant threat to human health and for which new treatments are urgently required. To guide and encourage research and development of new antibiotics, the list is defined according to the urgency of the need for new antibiotics as critical, high, and medium priority.

The majority of the pathogens on the WHO list are Gram-negative bacteria. Gram-negative bacteria are more resistant than Gram-positive bacteria because of their distinct structure and are responsible for significant morbidity and mortality worldwide. Several strategies have been described to combat and control resistant Gram-negative bacteria, including developing supplementary antimicrobial Agents, Structural modification of current antibiotics, and research and analysis of chemical structures with new modes of action and novel targets sensitive to resistant bacteria.

The ESCAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Clostridium difficile, Acinetobacter species, P. aeruginosa, and Enterobacteriaceae), which are organisms that can “escape” the effects of antimicrobial agents and cause the majority of hospital-acquired infections, are the most prevalent threats associated with antibiotic resistance. Noticeably, the last three are gram-negative organisms. 

Struggle Against Gram-Negative Bacterial Infections

The Secretary of State for Health has launched a critical ambition to reduce healthcare-associated Gram-negative bloodstream infections by 50% by 2021 and reduce inappropriate antimicrobial prescribing by 50% by 2021. Gram-negative bloodstream infections are believed to have contributed to approximately 5,500 NHS patient deaths in 2015. The number of cases reported by NHS trusts in England increased by 1.1% between 2016/17 and 2017/18 and 27.1% since 2012/13.

In most cases, GNBSIs are community-acquired (71%), occurring in older people and primarily through a UTI originating from the person’s bowel flora, Community-onset GNBSIs (71%) occur before hospital admission, with hospital-based onset (29%) occurring 48 hours after admission.

Many strategies and interventions have been implemented in England, significantly reducing methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections and Clostridium difficile infection (CDI). 

Novel ways to overcome intrinsic and acquired resistance in Gram-negative bacteria give hope for the future. Some treatments have shown effectiveness against Gram-negative resistant bacteria by deactivating the resistance mechanism, such as the action of β-lactamase inhibitor antibiotic adjuvants. Another interesting approach is the development of naturally derived antibacterial agents with antibacterial activity against novel targets, such as bacteriophages, which are bactericidal viruses that disrupt several bacterial functions.

Furthermore, strategies must be developed and implemented to reduce antibiotic use and unnecessary prescription. This can be implemented by

  1. Educational lectures, campaigns, or patient information handouts that explain antibiotic misuse and its consequences.
  2. Prevent self-medication by monitoring and recording antimicrobial medicine intake in pharmacies. This and other initiatives can improve patient education and reduce antibiotic misuse and prescription. 

In January 2019, the UK government published its vision for AMR to be contained and controlled by 2040.

The current national action plan focuses on three key ways of tackling AMR:

  • Reducing the need for, and unintentional exposure to, antimicrobials
  • Optimizing the use of antimicrobials
  • Investing in innovation, supply, and access


Resistance is growing among Gram-positive and Gram-negative bacteria, making it harder to make new antimicrobials. In the last decade, antibiotic drug development has slowed down, and there aren’t as many options for treating Gram-negative bacteria as there used to be. This makes the problem of antibiotic resistance a worldwide medical emergency. Even though there won’t be many new compounds on the market soon, antibiotic discovery is still one of the most effective ways to stop and reverse the rise of resistant bacteria. New antimicrobial classes and analogs of existing drugs need to be developed to nip the evil bud of AMR.