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Bacterial Pathogens

Bacterial Classification

Bacteria are classified within two major groups, Gram-positive or Gram-negative, based on their cell wall composition. Peptidoglycan is a main component of the cell wall that surrounds the cell membrane, provides protection from osmotic changes, and determines the bacterial cell shape. The rigid mesh-like peptidoglycan layer consists of two types of glycans, N-acetylglucosamine and N-acetylmuramic acid, connected by short peptides. In Gram-positive bacteria the peptidoglycan mesh is wider, consisting of multiple layers and associated teichoic acids, polysaccharides, and peptidoglycolipids. In contrast, Gram-negative bacteria have a thin cell wall with less peptidoglycan content and cross-linking. Gram-negative bacteria are additionally surrounded by an outer membrane containing lipopolysacharides (LPS) and lipoproteins.

Gram-Positive and Gram-Negative Bacteria

Gram staining allows bacterial classification based on the presence of a peptidoglycan containing cell wall.

Gram-Positive Bacterial Pathogens

Bacillus anthracis

Corynebacterium diphtheriae

Listeria monocytogenes

Staphylococcus aureus

Streptococcus pneumoniae

Clostridium botulinum 

Gram-Negative Bacterial Pathogens

Escherichia coli

Legionella pneumophila

Pseudomonas aeruginosa


Shigella dysenteriae

Vibrio cholerae

Bacterial envelope composition distinguishes two major bacterial groups. The Gram staining technique was developed by the Danish bacteriologist Hans Christian Gram. This technique identifies bacteria based on their cell wall composition and allows classification of most bacteria as Gram-positive or Gram-negative. Briefly, during Gram staining bacteria are exposed to crystal violet dye and iodine which results in the formation of a complex, crystal violet-iodine complex. A solvent step following dye treatment, dehydrates the thicker peptidoglycan layer trapping the dye and staining bacteria, thereby referred to as Gram-positive. In contrast, solvent treatment of bacteria with a thin peptidoglycan layer, dissolves the outer membrane and allows the dye to leach out, thereby referred to as Gram-negative.

Immunohistochemistry (IHC) of Streptococcus pneumoniae (pneumococcus) and influenza A-infected lung sections.

The pathogen, Streptococcus pneumoniae (pneumococcus), increases morbidity and mortality of influenza-infected hosts. Histopathology of lung sections collected at 24 h pbi from mice co-infected with mouse adapted influenza A/Puerto Rico/8/34 (H1N1) (PR8) and type 2 pneumococcal strain D39 variants. Serial lung sections were subjected to immunohistochemistry (IHC) for pneumococcus using Rabbit Anti-Streptococcus pneumoniae Polyclonal Antibody (Catalog # NB100-64502) at 4 µg/mL or neutrophils using Rat Anti-Ly-6G6C Monoclonal Antibody (Catalog # NB600-1387) at 2 µg/mL. Representative images at 4x magnification with 60x magnification inset are shown. Images courtesy of Dr. Amanda P. Smith, UTHSC, TN //

Analysis of Botulinum Neurotoxin Type A through solid phase sandwich ELISA.

Solid phase sandwich ELISA, Botulinum Neurotoxin Type A DuoSet ELISA, 5 plate (DY4489-05).  Botulinum neurotoxin is produced by Clostridium botulinum and consists of a light A- and heavy protein B-subunit. The A-subunit inhibits the release of acetylcholine at neuromuscular junctions.

Mycoplasma and Mycobacterium: How are they different?

Mycoplasma species are Gram-negative bacteria which lack a cell wall. The Mycoplasma genus contains over a 100 species, some of which lead to human disease including Mycoplasma pneumoniae and Mycoplasma genitalium. In contrast, Mycobaterium species are characterized by a complex cell wall, consisting of a mycolyl-arabinoglactan-peptidoglycan complex, which sets them apart from other Gram positive bacteria. The presence of an outer membrane “myco-membrane” is a feature that Mycobacterium species share with Gram negative bacteria. Close to 200 Mycobacterium species have been identified including important human pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae.

Western blot analysis of 65 kDa heat shock protein in Mycobacterium leprae’s cell lysate.

The 65-kDa heat shock protein (HSP65) is a predominant component of Mycobacterium leprae’s cell wall. HSP65 is highly immunogenic and recognized by T cells.Western blot analysis of the HSP65 expression in Mycobacterium leprae cell lysate using a mouse monoclonal antibody raised against Mycobacterium bovis BCG Hsp65, Hsp65 (mycobacterial) Antibody (4H11) [NBP1-97874].

Fluorescent Probes to Image Gram-Positive and Gram-Negative Bacteria

Incorporation of fluorescently labeled metabolic precursors by live bacteria results in the labeling of specific cell wall and envelope components such as peptidoglycans and trehalose glycolipids, respectively. In metabolic labeling, fluorescent reporters target specific cell surface structures allowing investigators to study bacterial growth and responses to pharmacological agents. Combined with super-resolution microscopy, fluorescent metabolic labeling enables the analysis of cell wall and envelope dynamics.

Fluorescent Probe Description Excitation/Emission Wavelength (nm)


Blue fluorescent D-amino acid, labels peptidoglycan in live bacteria.



Green fluorescent D-amino acid, labels peptidoglycan in live bacteria.



Orange-red fluorescent D-amino acid, labels peptidoglycan in live bacteria.



Green sulfonated BODIPY-FL 3-amino-D-alanine (sBADA), labels peptidoglycan in live bacteria.


6 TMR Tre

Fluorescent trehalose, selectively labels mycobacterial cell envelope.



Green-yellow lucifer yellow-based fluorescent D-amino acid, labels peptidoglycan in live bacteria.


Fluorescent probes available at Tocris:


Treatment of bacterial infections relies primarily on the use of antibiotics, which are chemicals that inhibit bacterial growth or kill bacteria. Based on their main mechanism of action, antibiotics are classified as bacteriostatic or bactericidal, depending on whether they stop bacterial growth or induce cell death, respectively. Beyond these major mechanisms, bactericidal antibiotics may induce cell death by targeting and inhibiting the synthesis of DNA, RNA, protein or cell wall components.

Antibiotic Description Mechanism

Gentamicin sulfate

Aminoglycoside antibiotic. Irreversibly binds bacterial ribosomes and disrupts protein synthesis. Active against gram-positive and -negative bacteria. Stable over wide range of temperatures.


Kanamycin sulfate

Broad spectrum antibiotic. Inhibits proteins synthesis. Binds bacterial 30S ribosomal subunit.



Aminoglycoside antibiotic. Exhibits antibacterial activity against strains of Enterobacteriaceae, Pseudomonas and Staphylococcus aureus.


Clindamycin hydrochloride

Lincosamide antibiotic. Inhibits protein synthesis by binding to the 50S ribosomal subunit and preventing early peptide chain elongation in bacteria.


Doxycycline hyclate

Broad-spectrum antibiotic. Tetracycline derivative. Inhibits bacterial protein synthesis. Binds bacterial 30S ribosomal subunit.


Antibiotics available at Tocris:

Find More Antibiotics from Tocris Bioscience, a Bio-Techne brand.

Antibiotic Resistance

Development of antibiotic resistant bacteria is currently a major public health concern. Antibiotic resistance mechanisms are diverse and encoded by bacterial genomic programs and extra-chromosomal or plasmid DNA. Several in vitro methods allow assessment of bacterial resistance to antibiotics including:

Disc diffusion testing- Involves culture of bacterial specimens on agar plates, followed by exposure to antibiotics through placement of discs containing antibiotic drug. Inhibition of growth around the antibiotic containing disc is used as a measure of antibiotic susceptibility.

Minimum inhibitory concentration- Involves inoculation of bacteria in serial dilutions of an antibiotic, typically broth, to determine the minimal antibiotic concentration which inhibits visible bacterial growth. This is based on the evaluation of bacterial growth following overnight culture.

Mechanisms of Antibiotic Resistance

Bacterial antibiotic resistance may be acquired by spontaneous genetic mutations or transfer of genetic material from other bacteria or bacteriophages. Various mechanisms of antibiotic resistance have been identified and bacteria may exhibit a single or multiple antibiotic resistance traits.

Antibiotic Resistance Mechanisms Bacterial Proteins

Enzyme mediated chemical modification reduces or abolishes antibiotic activity

Hydrolases (e.g., NDM-1); Transferases (e.g., Neomycin phosphotransferase II); Redox enzymes (e.g., Tetracycline monohydroxylase TetX)

Reduction of antibiotic uptake

Porins (aqueous channels, provide route for antibiotics into periplasmic space), mutations lead to reduced drug uptake (e.g., E. coli's OmpC, OmpF mutations and β-lactam resistance; Helicobacter pylori OMPs alterations and clarithromycin resistance)

Increased antibiotic efflux

Multidrug efflux pumps (e.g., Helicobacter pylori’s RND efflux pumps- HefABC, HefDEF, and HGHI; Methicillin resistance Staphylococcus aureus' NorB efflux pump overexpression) membrane proteins involve in extrusion of multiple types of antibiotics

Increased antibiotic target

Over-expression of antibiotic target leading to increased minimal inhibitory antibiotic concentration (e.g., E. coli dihydrofolate reductase over-expression leads to trimethoprim resistance).

Change in bacterial antibiotic target (e.g., mutations, recombination events)

Reduced or blocked antibiotic binding to key functional targets (e.g., Enterococcus vancomycin resistance due to decreased antibiotic binding to peptidoglycan precursors)

Major Bacterial Human Pathogens

In 2017 WHO published a list of "priority pathogens" which includes twelve bacterial families with increased pathogenic potential to humans due to their increased incidence of antibiotic resistance. The list classifies pathogens as critical, high and medium based on the global need for the development of new antibiotics.

*Priority Pathogens Antibiotic Resistance Description/Groups Affected
Critical Priority

Acinetobacter baumannii


Opportunistic pathogen found in water and soil, affects immunocompromised patients leading to infection (e.g., blood, urinary, and pneumonia).

Pseudomonas aeruginosa


Opportunistic pathogen found in water and soil, common in post-surgery associated infections (e.g., blood, and pneumonia).



Extended spectrum β-lactamase producing

Common infectious agent in hospital settings (e.g., E. coli, Klebsiella pneumoniae).

High Priority

Enterococcus faecium


Common infectious agent in hospital settings, frequently affects elderly and immunocompromised patients (e.g., urinary and blood infections).

Staphylococcus aureus

methicillin-resistant, vancomycin-intermediate and resistant

Community or health care associated infectious agent, spreads through contact with infected people or objects (e.g., skin infection through open wounds).

Helicobacter pylori


Frequently infects the stomach in an estimated 40% of people and may be asymptomatic or lead to peptic ulcers.

Campylobacter spp.


Common foodborne pathogen, infections usually occur through ingestion of contaminated foods (e.g., undercooked poultry, cross-contaminated foods).



Common foodborne pathogen, infections occur frequently in association with contaminated foods (e.g., raw or undercooked meats and eggs).

Medium Priority

Streptococcus pneumoniae


Upper respiratory tract commensal, may cause infection under conditions of host susceptibility (e.g., middle ear, lungs, blood, and meninges).

Haemophilus influenzae


Upper respiratory tract commensal, may cause infection under conditions of host susceptibility (e.g., middle ear, lungs, blood, and meninges).

Shigella spp.


Foodborne pathogen, infection results through contaminated foods and as the result of poor hand washing practices leading to intestinal disease.

*WHO priority pathogens list for research and development of new antibiotics

Immunocytochemical detection of adenovirus infected cells with anti-adenovirus mouse monoclonal antibody.

Quorum sensing inhibitors from Tocris allow modulation of bacterial communication by inhibiting the expression of virulence factors.

Helicobacter pylori binds to the gastric epithelium inducing inflammatory responses. Urase produced by Helicobacter pylori supports bacterial colonization and survival within the gastric mucosa. Urease also functions as a virulence factor and is implicated in the inflammatory process through various mechanisms (e.g., activation of CD74 in gastric epithelium and induction of IL8 production). A.Immunohistochemical analysis of Helicobacter pylori in Formalin-fixed paraffin-embedded human stomach tissue sections with a rabbit polyclonal antibody to Helicobacter pylori lysate, Helicobacter pylori Antibody [NBP2-29479]. B. Immunohistochemical staining of Helicobacter pylori urase B in tissue from mice gastric pylori with a rabbit polyclonal antibody, Helicobacter pylori urease B {NBP2-42850].

Anti-virulence Strategies to Target Multi-Drug Resistant Pathogens

Quorum sensing (QS) signaling is a mechanism of bacterial communication, which allows regulation of virulence factors, biofilm formation, colonization, and environmental adaptation. Bacterial communication is underscored by the release and extracellular accumulation of autoinducer molecules or AIs (e.g., autoinducer-2, acylated homoserine lactones (acyl-HSLs), oligopeptides, Pseudomonas quinolone signal molecule, diffusible signal factor, γ-butyrolactone, and 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde). Increase of extracellular AI concentration is dependent on bacterial density and leads to the activation of signaling pathways which regulate the expression of bacterial virulence genes. Targeting QS signaling has been proposed as a potential anti-bacterial strategy which may circumvent the development of bacterial resistance. Various strategies to inhibit QS depend on inhibition of AI expression or synthesis, inhibition of AI extracellular accumulation, and blockade of AI detection.

Explore Quorum Sensing Pathway

Quorum Sensing Inhibitors Description

Furanone C-30 (Cat. No. 6546)

Inhibits virulence factor expression in Pseudomonas aeruginosa. Increases bacterial susceptibility to antibiotics.

Ambuic acid (Cat. No. 6547)

Inhibits synthesis of autoinducer peptide in Methicillin-resistant Staphylococcus aureus.

Quorum sensing modulators available at Tocris:

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