Gram positive bacteria

Gram-positive bacteria form a major group of prokaryotes defined by their characteristic response to the Gram staining technique, a fundamental diagnostic method in microbiology. Organisms in this category possess a thick peptidoglycan layer in their cell walls, which enables them to retain the crystal violet–iodine complex during staining. When observed under the microscope, Gram-positive cells appear purple, in marked contrast to Gram-negative cells, which lose the primary stain during decolorisation and take up the counterstain, appearing pink or red. Although Gram staining is a practical and rapid means of classification, it does not always align with deeper evolutionary relationships, and modern genetic methods have refined understanding of the group.

Structural Features

The defining characteristic of Gram-positive bacteria is their cell envelope structure. Unlike Gram-negative organisms, which possess an inner membrane, a thin peptidoglycan layer, and an outer membrane, Gram-positive bacteria are monoderm, possessing only a single cytoplasmic membrane surrounded by a thick layer of peptidoglycan, typically 20–80 nanometres in thickness. This substantial wall not only confers rigidity but also forms the basis for the retention of the crystal violet stain.
Several key structural components are commonly present:

• Cytoplasmic membrane: a phospholipid bilayer that encloses the cytoplasm.
• Thick peptidoglycan layer: extensively cross-linked by the enzyme DD-transpeptidase, providing a rigid framework.
• Teichoic and lipoteichoic acids: polymers embedded within the cell wall that contribute to cell shape, immune interactions, and ion regulation.
• Surface layer (S-layer): a crystalline protein or glycoprotein lattice attached directly to the peptidoglycan.
• Capsule (in some species): typically polysaccharide-based, offering protection and enhancing pathogenicity.
• Flagella (when present): supported by a two-ring basal structure, distinguishing them from the four-ring apparatus of Gram-negative bacteria.
• Minimal periplasm: although a narrow space exists between membrane and wall, it is far smaller than in diderm organisms.

These features collectively influence staining characteristics, antibiotic susceptibility patterns, and interactions with host tissues.

Gram Staining and Diagnostic Use

Gram-positive bacteria retain the crystal violet–iodine complex during the decolorisation step because their thick, multilayered peptidoglycan prevents the dye from being washed out. In contrast, Gram-negative bacteria lose the dye due to the dissolution of their outer membrane and increased permeability during alcohol treatment. As a result, Gram-positive bacteria are especially susceptible to antibiotics that target cell wall synthesis, such as β-lactams, although resistance mechanisms exist in several clinically important species.
Gram staining remains valuable for the rapid identification of pathogens in clinical samples. For example, in cerebrospinal fluid specimens, Gram-positive cocci can be distinguished from Gram-negative rods and from host immune cells, providing immediate guidance for treatment decisions in conditions such as meningitis.

Classification and Phylogeny

Traditional bacterial classification relied heavily on Gram staining, leading to the establishment of categories such as Bacillota (then considered Gram-positive), Gracilicutes (Gram-negative), Mollicutes, and Mendocutes. However, advances in molecular phylogenetics, particularly analyses of 16S ribosomal RNA sequences, revealed that Gram-positive bacteria are not always monophyletic as a staining-defined group. They separate into two major evolutionary lineages based on their genomic guanine–cytosine content:

• High G+C group (Actinomycetota): includes genera such as Corynebacterium, Mycobacterium, Nocardia, and Streptomyces, noted for complex cell envelopes and diverse metabolic capabilities.
• Low G+C group (Bacillota): includes Bacillus, Clostridium, Staphylococcus, Streptococcus, Enterococcus, and related genera.

Although both lineages share the monoderm cell envelope architecture, their evolutionary divergence is strongly supported by genomic evidence.

Cell Envelope Structure and Evolutionary Considerations

While Gram staining provides an empirical means of grouping bacteria, the classification conflates staining response, envelope architecture, and taxonomic lineage, which do not always coincide. Gram-positive bacteria are defined structurally as monoderm organisms with thick peptidoglycan layers. Some organisms that retain the Gram stain are not monoderm, and others that are monoderm may stain Gram-negative for compositional reasons.
The monoderm architecture is widely regarded as ancestral in bacteria. Evidence for this includes the sensitivity of monoderm organisms to many antibiotics and the widespread production of antibiotic substances among Gram-positive bacteria. Diderm (Gram-negative) bacteria appear to have evolved an outer membrane as a protective adaptation, conferring resistance to environmental stress and antimicrobial compounds.
Several bacterial groups demonstrate intermediate or exceptional features:

• Deinococcota: stain Gram-positive yet possess a diderm envelope, illustrating transitional characteristics.
• Mycoplasmas: lack peptidoglycan entirely, rendering Gram staining ineffective, despite their evolutionary relationship to monoderm taxa.
• Negativicutes, Fusobacteriota, Synergistota, and Elusimicrobiota: members of these groups possess diderm structures despite clustering phylogenetically within or near traditional Gram-positive phyla.
• Chloroflexota: generally monoderm, although staining reactions vary among species.

Conserved molecular signatures, such as specific insertions in the HSP60 (GroEL) protein, help delineate true LPS-containing Gram-negative phyla from atypical diderm groups and from the traditional monoderm lineages.

Major Gram-Positive Groups and Notable Examples

Gram-positive bacteria encompass a broad array of habitats, morphologies, and physiological roles. Several medically and industrially important groups include:

• Staphylococci: Gram-positive cocci arranged in clusters; Staphylococcus aureus is a major cause of skin infections, pneumonia, and septicaemia.
• Streptococci and enterococci: cocci forming chains or pairs; associated with pharyngitis, endocarditis, and urinary infections.
• Bacilli and clostridia: rod-shaped organisms; Bacillus anthracis and Clostridium botulinum exemplify important pathogens.
• Actinomycetes: filamentous soil organisms; Streptomyces species are notable antibiotic producers, and Mycobacterium includes the agents of tuberculosis and leprosy.

These organisms differ widely in ecology and metabolic diversity but share the common monoderm envelope structure and the capacity to retain the Gram stain.

Exceptions, Variability, and Diagnostic Limitations

Not all bacteria conform neatly to the Gram-positive/Gram-negative dichotomy. Several exceptions arise due to variations in cell wall composition, the presence or absence of an outer membrane, or unique structural components such as mycolic acids.
Some clinically relevant taxa exhibit atypical staining behaviour:

• Mollicutes: lack cell walls and do not retain crystal violet.
• Mycobacteriales: display acid-fast properties due to their waxy, mycolic acid–rich outer layers; they require alternative staining methods such as Ziehl–Neelsen.
• Negativicutes: stain Gram-negative despite phylogenetic placement within Bacillota.
• TM7 and Ktedonobacteria: monoderm taxa with variable staining responses.