GRAM POSITIVE AND NEGATIVE BACTERIA AND MYCOBACTERIUM SPECIES

Lesson Objectives
By the end of this lesson, students will be able to:
1. Explain gram-negative and positive bacteria structures
2. Compare and contrast gram-positive and negative stain
3. Name the causative agents for tuberculosis, non-tuberculous mycobacterial
infections, and leprosy.
4. Define "acid fastness" and describe the principle of acid-fast staining.
5. Describe the process of infection leading to active primary and secondary
tuberculosis.
6. Explain how tuberculosis is diagnosed.
7. What are the characteristics of M. leprae and M. ulceran

GRAM POSITIVE AND NEGATIVE BACTERIA

Gram-negative Bacteria
Gram-negative bacteria are bacteria that do not retain the crystal violet dye in the Gram stain
protocol. Gram-negative bacteria will thus appear red or pink following a Gram stain procedure
due to the effects of the counterstain (for example safranin). Gram-negative bacteria have a
cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane containing
lipopolysaccharide. There is a space between the cytoplasmic membrane and the outer
membrane called the periplasmic space or periplasm. The periplasmic space contains the loose
network of peptidoglycan chains referred to as the peptidoglycan layer.
Examples: Acinetobacter, Actinobacillus, Bordetella, Brucella, Campylobacter, Cyanobacteria,
Enterobacter, Escherichia coli, Franciscella, Helicobacter, Hemophilus, Klebsiella, Legionella,
Moraxella, Neisseria, Pasteurella, Proteus, Pseudomonas, Salmonella, Serratia, Shigella,
Treponema, Vibrio &Yersinia
Gram-positive bacteria retain the color of the crystal violet stain in the Gram stain. This is
characteristic of bacteria that have a thick cell wall composed of a thick layer of a particular
substance (called peptidologlycan).

Examples: Streptococcus and Staphylococcus, are cocci (sphere-shaped). The

remaining organisms are bacilli (rod-shaped) and can be subdivided based on their ability to
form spores. The non-spore formers are Corynebacterium and Listeria (a coccobacillus),
whereas Bacillus and Clostridium produce spores.

Infections gram-positive: Staphylococcal Infections. Staphylococci are gram-positive

aerobic organisms. Staphylococcus aureus is the most pathogenic; it typically causes skin
infections and sometimes pneumonia, endocarditis, and osteomyelitis. It commonly leads
to abscess formation Antibiotics used to treat gram-negative bacteria: These antibiotics
include cephalosporins (ceftriaxone-cefotaxime, ceftazidime, fluoroquinolones (ciprofloxacin,
levofloxacin), aminoglycosides (gentamicin, amikacin), imipenem, broad-
spectrum penicillins with or without β-lactamase inhibitors (amoxicillin-clavulanic
acid, piperacillin-tazobactam),

Infection Gram-negative: UTIs, diarrhea, peritonitis, and bloodstream infections are commonly
caused by gram-negative bacilli. Gram-negative bacteria cause plague, cholera, and typhoid
fever. Antibiotic used to treat gram-positive bacteria: Antibiotics for Gram-Positive
Bacterial Infection: Vancomycin, Teicoplanin,
Quinupristin/Dalfopristin, Oxazolidinones, Daptomycin, Telavancin, and Ceftaroline.

1. There is a membrane present around the cell wall of gram-negative bacteria which
increases the risk of toxicity to the host but this membrane is absent in gram-positive
bacteria.
2. Porin channels are present in gram-negative bacteria which can prevent the entry of
harmful chemicals and antibiotics like penicillin. These channels can also expel out
antibiotics making much more difficult to treat in comparison to gram-positive bacteria.
3. The risk of resistance against antibiotics is more in Gram-negative bacteria due to the
presence of external covering around the cell wall.
4. Gram-negative bacteria possess both exotoxins and endotoxins but in the case of
gram-positive bacteria there are only exotoxins.

Gram-negative Bacteria versus Gram-positive Bacteria comparison chart

Gram-negative Bacteria Gram-positive Bacteria

Gram reaction Can be decolorized to accept Retain crystal violet dye and stain dark
 Gram-negative Bacteria versus Gram-positive Bacteria comparison chart

Gram-negative Bacteria Gram-positive Bacteria

counterstain (Safranin or neutral red); violet or purple, they remain colored

stain red or pink, they don't retain the blue or purple with gram stain when
Gram stain when washed with absolute washed with absolute alcohol and
alcohol and acetone. water.

Peptidoglycan layer Thin (single-layered) Thick (multilayered)

Teichoic acids Absent Present in many

Periplasmic space Present Absent

Outer membrane Present Absent

Lipopolysaccharide High Virtually none

(LPS) content

Lipid and High (due to presence of outer Low (acid-fast bacteria have lipids
lipoprotein content membrane) linked to peptidoglycan)

Flagellar structure 4 rings in basal body 2 rings in basal body

Toxins produced Primarily Endotoxins Primarily Exotoxins

Resistance to Low High

physical disruption

Inhibition by basic Low High

dyes

Susceptibility to Low High

anionic detergents

Resistance to sodium Low High

 Gram-negative Bacteria versus Gram-positive Bacteria comparison chart

Gram-negative Bacteria Gram-positive Bacteria

azide

Resistance to drying Low High

Cell wall The cell wall is 70-120 Å (ångström) The cell wall is 100-120 Å thick; a
composition thick; two-layered. Lipid content is 20- single layer. Lipid content of the cell
30% (high), Murein content is 10-20% wall is low , whereas Murein content is
(low). 70-80% (higher).

Mesosome Mesosome is less prominent. Mesosome is more prominent.

Antibiotic More resistant to antibiotics. More susceptible to antibiotics

Resistance

MYCOBACTERIUM
Are non-motile acid-fast bacilli that don’t form spore or capsule belongs to genus
mycobacterium. They are about 40 species that are accepted by the international committee of
systemic Bacteria. Only a few of this are pathogen other are saprophytes of little medical
importance
Medical importance
 I. Obligate pathogens –M. tuberculosis- Tuberculosis

-M. Africanum
- M. bovis
- M. ulcerans
- M. leprae - Leprosy
- M. ulceran – Skin ulcers

Opportunistic pathogens – M. kansasii

M. malmoense mainly pulmonary infection
M. szulgai
M. xenopi
M. avium
M. scrofulaceum Cervical adenitis
M. fortuitum
M. chelonei Skin infection
M. marinnum

Acid Fastness of mycobacterium

Importance characteristic of mycobacterium mean which stains with carbol funshin in Ziel-
Neelsen techniques they are able to retain or hold fast to their red color even when wash with
an acid solution other organisms are discolorise. Mycobacterium species varies in their degree of
acid fastness e.g. M. leprae is less acid-fast than M. tuberculosis hence require weaker acid
decolorize. Acid fastness is caused by a thick wall composed of waxes and lipid which has a
high content of mycolic fatty acid and this cell wall making it difficult to stain mycobacterium
with other techniques than Niel Neelsen techniques. Stain poorly with gram stain, stain gram +ve
or not stain at all. In a culture with exception of M. leprae can grow aerobically using protein-
enriched culture media with an atmosphere of increased CO 2 increased the growth of
mycobacterium.

M. tuberculosis

Habitat
The reservoir is an infected human but can also occur as a pathogen in an animal. It’s transmitted
by the infected person by coughing and spitting then inhaled by others. M. bovis with close
contact with infected cattle or infected milk, Bovis strain can be transmitted from a person but
the incident is low compare to M. tuberculosis.
Pathogenicity
About 10 million contact tuberculosis in the developing world and 3 million, they are
multiplication in the lung and near the lymph gland, and in children, they are enlarging in the
lymph gland. This first infection is called primary tuberculosis infection. Most cases of the
recovery from this infection leaving the infected area calcified mostly in the lungs. Reactivation
may occur due to poor health, malnourished, or underlying immune infection. Infection can
spread through the lymphatic system and blood circulation to the lungs and pleural cavity,
Kidney, bone, and joints

Features of different tuberculosis

i. Pulmonary tuberculosis
In the lungs, it caused inflammation leading to liquefied destruction of the lung with a
breakdown of diseased tissue into a cheese-like mass. The yellow pieces of material (sputum)
contain a lot of bacillus cough by the patient and difficulty in breathing is due to the developing
of a cavity in the lungs. Symptoms are: chronic cough with production of mucopurulent sputum
and this may contain blood. In later stages they are loss of weight, fever, sweating especially
when sleeping, tiredness, chest pain, and anemia resulting in complications such as lung
collapsed. In children it’s difficult to diagnose because they rarely do not cough with sputum
production, Diagnosis can be using a positive tuberculin reaction.

ii. Renal/urinogenital TB
Infection may reach the kidney through blood circulation and this causes the production of pus
cells in urine. No organism can be isolated in urine culture in later stages they are frequently
passing of urine, reoccurring fever this may cause infertility and pelvis inflammatory.

iii. Miliary tuberculosis

Here patients become acutely heal with fever it’s recognized with nodules on chest x-ray. It may
be accompanied by low cell count and may lead to leukemia especially in the elderly. The
spleen, liver, lymph gland become enlarge and meninges may be affected.

iv. Tuberculous meningitis

When bacilli reach meninges due to blood circulation more frequently in infant and young
children, this condition is fatal unless the treat at the early stage, it requires cerebrospinal fluid
(CSF) to be examined for acid-fast bacilli.

Laboratory Diagnosis

The microbiological diagnosis of tuberculosis (TB) is an important tool for disease control. It
consists of both conventional methods (acid-fast microscopy, culture, biochemical identification,
anti-tuberculosis drug-susceptibility testing; DST) and modern molecular techniques
Treatment and prevention: In children by giving BCG vaccines at an early stage but adult
require drug with anti TB drugs.

MYCOBACTERIUM LEPRAE
It causes leprosy
Habitat
From the nose and urinary tract of peoples, Bacteria may be inhaled or enter the skin. How
is leprosy transmitted? ... Researchers suggest that M. leprae are spread person to person by
nasal secretions or droplets. However, the disease is not highly contagious like the flu. They
speculate that infected droplets reach other peoples' nasal passages and begin the infection there

Pathogenicity
Leprosy (Hansen's disease) is a chronic infectious disease that primarily affects the peripheral
nerves, skin, upper respiratory tract, eyes, and nasal mucosa (lining of the nose).
The disease is caused by a bacillus (rod-shaped) bacterium known as Mycobacterium leprae.
Group into five based on cellular immune responses.

Type of leprosy Symbol Immune response

- Tuberculoid TT Good
- Borderline tuberculoid BT Not very
good
- Mid-Borderline BB Partial
- Borderline lepromatus BL poor
- Lepromatous LL Nil

Leprosy is defined by the number and type of skin sores you have. Specific symptoms and treatment
depend on the type of leprosy you have. The types are:

Tuberculoid. A mild, less severe form of leprosy. People with this type have only one or a few
patches of flat, pale-colored skin (paucibacillary leprosy). The affected area of skin may feel numb
because of nerve damage underneath. Tuberculoid leprosy is less contagious than other forms.

Lepromatous. A more severe form of the disease. It has widespread skin bumps
and rashes (multibacillary leprosy), numbness, and muscle weakness. The nose, kidneys, and male
reproductive organs may also be affected. It is more contagious than tuberculoid leprosy.
Borderline. People with this type of leprosy have symptoms of both the tuberculoid and lepromatous
forms

What Are the Symptoms of Leprosy?

Leprosy primarily affects the skin and the nerves outside the brain and spinal cord, called the
peripheral nerves. It may also strike the eyes and the thin tissue lining the inside of the nose.

The main symptom of leprosy is disfiguring skin sores, lumps, or bumps that do not go away after
several weeks or months. The skin sores are pale-colored.

Nerve damage can lead to:

 Loss of feeling in the arms and legs

 Muscle weakness
It usually takes about 3 to 5 years for symptoms to appear after coming into contact with the leprosy-
causing bacteria. Some people do not develop symptoms until 20 years later. The time between
contact with the bacteria and the appearance of symptoms is called the incubation period. Leprosy's
long incubation period makes it very difficult for doctors to determine when and where a person with
leprosy got infected.

How Is Leprosy Diagnosed?

If you have a suspicious skin sore, your doctor will remove a small sample of the abnormal skin and
send it to a lab to be examined. This is called a skin biopsy. A skin smear test may also be done.
With paucibacillary leprosy, no bacteria will be detected. In contrast, bacteria are expected to be
found on a skin smear test from a person with multibacillary leprosy.

How Is Leprosy Treated?

Treatment depends on the type of leprosy that you have. Antibiotics are used to treat the infection.
Long-term treatment with two or more antibiotics is recommended, usually from six months to a
year. People with severe leprosy may need to take antibiotics longer. Antibiotics cannot treat nerve
damage.

Anti-inflammatory drugs are used to control nerve pain and damage related to leprosy. This may
include steroids, such as prednisone.

Patients with leprosy may also be given thalidomide, a potent medication that suppresses the body's
immune system. It helps treat leprosy skin nodules. Thalidomide is known to cause severe, life-
threatening birth defects and should never be taken by women who are pregnant or women who may
become pregnant.

Leprosy Complications
  Blindness or glaucoma.
 Disfiguration of the face (including permanent swelling, bumps, and lumps).
 Erectile dysfunction and infertility in men.
 Kidney failure.
 Muscle weakness that leads to claw-like hands or an inability to flex the feet.
 Permanent damage to the inside of the nose, which can lead to nosebleeds and a chronic,
stuffy nose.
 Permanent damage to the nerves outside the brain and spinal cord, including those in the
arms, legs, and feet.

MYCOBACTERIUM ULCERAN
Cause boruli ulcer
Habitat
It inhabits the soil and swampy area it may enter human through damage skin, cut and insect bite
wound.
Causative organism
Mycobacterium ulcerans grows at temperatures between 29–33 °C (Mycobacterium
tuberculosis grows at 37°C) and a low 2.5% oxygen concentration to grow. The organism
produces a unique toxin – mycolactone – which causes tissue damage and inhibits the immune
response.
Transmission
The exact mode of transmission for M. ulcerans is still unknown.
Signs and symptoms
Buruli ulcer often starts as a painless swelling (nodule). It can also initially present as a large
painless area of induration (plaque) or diffuse painless swelling of the legs, arms, or face
(edema). Local immunosuppressive properties of the mycolactone toxin enable the disease to
progress with no pain and fever. Without treatment or sometimes during antibiotics treatment,
the nodule, plaque, or oedema will ulcerate within 4 weeks with the classical, undermined
borders. Occasionally, bone is affected causing gross deformities.
Laboratory: Polymerase chain reaction (PCR), direct microscopy, histopathology, and culture.
PCR is the most commonly used method.
Treatment: Treatment consists of a combination of antibiotics and complementary treatments
(under morbidity management and disability prevention/rehabilitation). Treatment guidance for
health workers can be found in the WHO publication "Treatment of mycobacterium ulcerans
disease (Buruli ulcer)."
Quiz
1. Describe why it is difficult to treat gram-negative bacteria in comparison to gram-
positive bacteria (6mks)
2. Describe the different gram-positive and negative bacteria structures 7mks
3. Explain the meaning of acid fastness mycobacterium 4mks
4. Describe types of leprosy 6mks
5. Lists complications associated with leprosy 5mks

References

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stainingprocedures.p 536. In Jorgensen JH, Pfaller MA, Carroll KC, Funke G, Landry M
L, Richter SS, Warnock DW (ed), Manual of clinical microbiology, 11th ed, vol 1. ASM
Press, Washington, DC.

2. Tortoli E. 2012. Phylogeny of the genus Mycobacterium: many doubts, few

certainties. Infect Genet Evol 12:827–831. doi: 10.1016/j.meegid.2011.05. 025.

3. Tortoli E. 2014. Microbiological features and clinical relevance of new species of the
genus Mycobacterium. Clin Microbiol Rev 27:727–752. doi:10.1128/CMR.00035-14.

4. World Health Organization. 2017. Leprosy elimination: WHO recommended MDT

regimens. World Health Organization, Geneva,
Switzerland. http://www.who.int/lep/mdt/regimens/en/. Accessed 21 September 2017.

5. World Health Organization. 2017. Buruli ulcer (Mycobacterium ulcerans infection)

fact sheet. World Health Organization, Geneva,
Switzerland. http://www.who.int/mediacentre/factsheets/fs199/en/. Accessed 21
September 2017