Gram-Negative - Gram bacteria -

Introduction

Gram-negatives are bacteria that - after being subjected to the Gram staining technique - take on a color ranging from pink to red.

Gram staining is a method used to classify bacteria according to the characteristics of their cell wall. Belonging to the same group - Gram-negative or positive - does not mean that there is a phylogenetic relationship between the various bacterial species of that group.

Bacterial cell wall

The bacterial cell wall can be defined as a rigid structure that encloses the bacterial cell giving it a certain robustness and conditioning its shape.

The fundamental element that constitutes the bacterial cell wall is peptidoglycan (otherwise known as bacterial or murein mucopeptide ).

Peptidoglycan is a polymer consisting of long linear polysaccharide chains, joined together by cross-links between amino acid residues.

The polysaccharide chains are composed by the repetition of a disaccharide, consisting in turn of two monosaccharides: the N-acetylglucosamine (or NAG ) and the N-acetylmuramic acid (or NAM ), linked together by β-1 type glycosidic bonds, 6.

The disaccharides are then bonded to each other with β-1, 4 type glycosidic bonds.

Linked to each molecule of NAM we find a "tail" of five amino acids (a pentapeptide ) that ends with two amino acids equal, more precisely, with two molecules of D-Alanine .

It is these terminal D-Alanine molecules that - following the action of the enzyme transpeptidase - allow the formation of cross-links between the parallel chains of the peptidoglycan.

More specifically, the transpeptidase originates a peptide bond between the third amino acid of a polysaccharide chain and the fourth amino acid of the parallel polysaccharide chain.

Cell wall functions

The bacterial cell wall plays a very important protective role against the bacterial cell, but not only, it is also able to regulate the transport of substances within the cell itself.

Therefore, it can be stated that the main functions of the cell wall are:

Prevent the breakdown of bacterial cells due to osmotic pressure. In fact, very often, bacteria live in hypotonic environments, that is in environments in which large quantities of water are present and which are "more diluted" than the internal environment of the bacterial cell. This difference in concentration causes the water to pass from the external environment (less concentrated) into the bacterial cell (more concentrated) in an attempt to equalize the concentration between the two environments. The uncontrolled entry of water would cause the bacterial cell to swell until it burst (osmotic lysis).
The function of the cell wall is precisely to resist the external pressure of the water, thus preventing swelling and bacterial lysis.
Protect the plasma membrane and the cellular environment from molecules or substances harmful to the same beat.
Adjust the entry of nutrients into the bacterial cell.

All that has been described so far is valid both for the Gram-negative cell wall and for the Gram-positive cell wall.

However, since the purpose of this article is to provide indications on the characteristics of Gram-negative bacteria, only the cell wall of the latter will be described below and that of Gram-positives will not be considered.

Gram-negative cell wall

In the Gram-negative wall the peptide bond formed between the polysaccharide chains of the peptidoglycan is direct.

The Gram-negative cell wall is very thin and has a thickness of 10 nm, but it is rather complex, since the peptidoglycan is surrounded by an external membrane anchored to it.

The outer membrane consists of an inner phospholipid leaflet and an outer leaflet formed by lipopolysaccharide (or LPS ).

The outer membrane and the peptidoglycan are linked together through lipoproteins . Since the presence of only lipoproteins on the outer membrane would hinder the passage of hydrophilic molecules, other particular protein complexes called porins are also present on the membrane. Porines are channels that allow the passage of small hydrophilic molecules.

For the transport of larger molecules, however, there are other carrier proteins, the carriers .

The space present between the outer membrane and the peptidoglycan is called periplasm and contains proteins and enzymes with biological functions.

Lipopolysaccharide is replaced by three distinct parts:

An internal lipidic part called lipid A which has endotoxin functions, therefore plays an important role in the pathogenicity of Gram-negatives;
A central polysaccharide portion called core ;
An external polysaccharide chain called the antigen O. This polysaccharide consists of simple sugars of various kinds, combined in blocks of three or five units and repeated several times to form molecules with certain antigenic characteristics typical of each bacterial species.

Gram stain

Gram staining is a procedure devised and developed in 1884 by Danish bacteriologist Hans Christian Gram.

The first stage of this procedure involves the preparation of a hot smear (ie a thin film of the material to be analyzed). In other words, a sample of the bacteria to be analyzed is placed on a slide and - through the use of heat - the micro-organisms are killed and blocked on the slide itself (hot fixation). After preparing the smear, you can proceed with the actual coloring.

The Gram staining technique involves four main phases.

Phase 1

The hot-fixed smear must be covered with the crystal violet dye (also known as gentian violet) for three minutes. In this way all the bacterial cells will turn purple.

Step 2

At this point, Lugol's solution (an aqueous solution of iodine and potassium iodide, defined as an etching agent as it is able to fix the color) is poured onto the slide and it is left to act for about a minute.

Lugol's solution is polar and penetrates the bacterial cell where it meets the crystal violet with which it forms a hydrophobic complex.

Step 3

The slide is washed with a bleach (usually, alcohol or acetone) for about twenty seconds. Then wash it with water to stop the action of the bleach.

At the end of this phase, the Gram-positive bacteria cells will have retained the purple color.

Gram-negative cells, on the other hand, will be discolored. This happens because the alcohol attacks the lipopolysaccharidic structure of the outer membrane of these bacteria, thus facilitating the loss of the previously absorbed dye.

Step 4

A second dye (usually, acid fuchsin or safranin ) is added to the slide and a couple of minutes is left to act.

At the end of this phase, the cells of Gram-negative bacteria, previously discolored, will take on a color ranging from pink to red.

Types of Gram-negative bacteria

Like the Gram-positive group, the Gram-negative group also includes numerous bacterial species.

In the following, some of the main bacteria belonging to this group will be briefly illustrated.

Escherichia coli

E. coli is a bacteria normally present in human intestinal bacterial flora, but in immunocompromised subjects it can give rise to opportunistic infections.

In fact, E. coli is responsible for opportunistic infections that cause diseases such as urethrocystitis, prostatitis, neonatal meningitis, enterohemorrhagic colitis, watery diarrhea or traveller's diarrhea or sepsis.

Depending on the type of infection that E. coli triggers, different types of antibiotics can be used. The most used drugs are carbapenems, some penicillins, monobactams, aminoglycosides, cephalosporins or macrolides (such as clarithromycin or azithromycin).

Bacteria belonging to the genus Salmonella

These bacteria are responsible for infections of the gastrointestinal tract that can cause diseases such as gastroenteritis, typhoid (enteric fever) and diarrhea.

To combat infections caused by these bacteria, ciprofloxacin, amoxicillin or ceftriaxone are usually used.

Klebsiella pneumoniae

K. pneumoniae is responsible for genito-urinary tract infections that cause cystitis, prostatitis or urethrocystis and respiratory infections that cause lung abscesses or pneumonia.

Cephalosporins, carbapenems, fluoroquinolones or some types of penicillins are used to treat infections with K. pneumoniae .

Bacteria belonging to the genus Shigella

These microorganisms are responsible for the onset of diseases such as bacillary dysentery and acute gastroenteritis.

Usually, fluoroquinolones are used to treat this type of infection.

Vibrioni (or Vibrio)

The vibrios are curved bacilli, that is, bacteria characterized by a "comma" shape.

Among the pathogenic vibes for man, we recall:

Vibrio cholerae, responsible for the onset of cholera. Generally, V. cholerae infections are treated with tetracycline or fluoroquinolones.
Vibrio parahaemolyticus, responsible for gastroenteritis, enterocolitis, diarrhea and dysenteric-like syndrome.

In case of infection with V. parahaemolyticus antibiotics such as fluoroquinolones or tetracyclines may be used. In some cases antibiotic therapy can be avoided and a symptomatic treatment can be performed.

Bacteria belonging to the genus Yersinia

The bacteria of the genus Yersinia are bacilli, that is, they are bacteria characterized by a cylindrical shape.

Among the Yersinia pathogens for humans, we recall:

Yersinia enterocolitica, responsible for the onset of gastrointestinal infections that cause diseases such as acute gastroenteritis or mesenteric adenitis. Y. enterocolitica infections are usually treated with antibiotics such as fluoroquinolones, sulfonamides or aminoglycosides.
Yersinia pestis, responsible for the onset of bubonic plague. Infections caused by Y. pestis can be treated with aminoglycosides, chloramphenicol or fluoroquinolones.

Campylobacter jejuni

C. jejuni is a spiral-shaped bacillus responsible for the onset of acute enteritis and diarrhea.

The infections he causes can be treated with macrolides (such as, for example, erythromycin) or with fluoroquinolones.

Helicobacter pylori

H. pylori is a curved bacillus responsible for the onset of gastrointestinal diseases such as chronic active gastritis and peptic ulcer.

The treatment for the eradication of Helicobacter pylori involves the use of three different types of drugs:

Colloidal bismuth, a cytoprotective used to prevent the adhesion of Helicobacter pylori to the gastric mucosa;
Omeprazole or another proton pump inhibitor so as to reduce acid secretion of the stomach;
Amoxicillin and / or clarithromycin, tetracycline or metronidazole (antibiotic drugs to kill bacterial cells).

Haemophilus influenzae

H. influenzae is a Gram-negative bacterium responsible for respiratory tract and nervous system infections that can cause acute otitis, epiglottitis, sinusitis, bronchitis, pneumonia or acute bacterial meningitis.

The antibiotics commonly used to treat H. influenzae infections are cephalosporins, penicillins or sulfa drugs.