GRAM-POSITIVE BACTERIAL INFECTIONS OF

VETERINARY IMPORTANCE

ASSOC. PROF. DR. BASIT ZESHAN

DVM, MPhil, PhD (NJAU, CHINA)
 Virulence Factor Terms

 Invasins:
 enabling bacterial entry into the cell by phagocytosis. By entering the
cytoplasm of the host cell, it has a ready supply of nutrients and is able to
protect the bacteria from complement, antibodies, and certain other body
defenses.
 Adhesins:
 surface proteins found in the cell wall of various bacteria to enable them to
bind to specific receptor molecules on the surface of host.
 Virulence Factor Terms

 Enterotoxin: acts on the intestinal wall (causes GI upset)

◦ tend to be produced by Gram-positive bacteria rather than by Gram-
negative bacteria.
◦ Endotoxin: Released by dead bacteria mainly (gram-negative bacteria)
◦ Lipopolysaccharide (LPS): a protein in the cell wall of many Gram
negative organisms
◦ Lipid A: A portion of the lipopolysaccharide which is also antigenetic
 Exotoxin: produced by a bacterium and then released from the cell into the
surrounding environment. The damage caused by an exotoxin can only occur
upon release.
◦ Hemolysins: cause rupture of red blood cells
 Neurotoxin: disrupts nerve cells
 Virulence Factor Terms
 H Ag (flagella)
 K Ag: an antigenetic protein on the capsule
 O Ag: a string of sugars on the lipopolysaccharide (LPS)in bacterial cell walls.
 β lactamase: blocks the ability of certain antibiotics (penicillin) to destroy the
bacteria
 MDR plasmids (genes that provide tetracycline resistance)
 Hyaluronidase: dissolves fluid between cells so bacteria can spread faster
between tissue planes
 SOD (superoxide dismutase): enzyme that deactivates contents of lysosomes.
 Staphylokinase: digests clots so bacteria can spread
 Coagulase: prevents blood coagulation so organism can spread
Gram-positive Cell Structure and Pathogenicity

 Morphologically,
gram-positive bacteria are composed of a cell wall, a single
cytoplasmic membrane, and cytosol.

 The cell wall is a thick, coarse structure that serves as an exoskeleton. Buried
within the cell wall are enzymes called transpeptidases, commonly referred
to as penicillin-binding proteins (PBPs).

 PBPs are a group of enzymes responsible for the building and maintenance
of the cell wall.
PBPs normally catalyze the cross-linking of the bacterial cell wall, but they can be
permanently inhibited by penicillin and other β-lactam antibiotics. (NAM = N-
acetylmuramic acid; NAG = N-acetylglucosamine
 Other protective mechanisms: outer capsule or biofilm that extends beyond
the cell wall itself and interfaces with the external milieu.
 Hydrolase enzymes located within the cytoplasmic membrane, called β-
lactamases, serve a protective role for the bacteria.
 Once attacked by the hydrolases, the β-lactam antibiotics are no longer
capable of binding to PBPs in normally susceptible bacteria.

 Peptidoglycan is the basic structural component of the cell wall of gram-

positive bacteria, accounting for 50% to 80% of the total cell wall content.
 Like endotoxin, peptidoglycan is released by bacteria during infection,
reaches systemic circulation, and exhibits proinflammatory activity.
 Lipoteichoic acids found in the gram-positive cell wall have both structural
and epithelial adherence functions. Lipoteichoic acid induces a
proinflammatory cytokine response, the production of nitric oxide, and may
lead to cardiovascular compromise.

 In addition to structural components, gram-positive organisms produce

soluble exotoxins that may play a role in the pathogenesis of sepsis.

 Much attention is focused on the roles of super-antigenic exotoxins that

promote the massive release of cytokines, potentially leading to shock and
multiorgan failure in both human and veterinary patients
Figure. Super antigens induce T cell activation by preventing conventional
antigen presentation pathways
STAPHYLOCOCCUS

 Normal members of every human’s microbiota

 Can be opportunistic pathogens
 Gram-positive cocci, nonmotile, facultative anaerobes
 Cells occur in grapelike clusters because cells division occurs
along different planes and the daughter cells remain attached to
one another
 Salt-tolerant-allows them to tolerate the salt present on human
skin
 Tolerant of desiccation-allows survival on environmental
surfaces (fomites)
 Classification

Family Staphylococcaceae
Genus Staphylococcus
Species S. aureus; S. intermedius; S. epidermidis
S. hyicus, S. canis
Differentiating Staphylococcus from Streptococcus

 The staphylococci produce the enzyme catalase that distinguishes them from
the streptococci which do not

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TOXING AND ENZYMES

 Exotoxins

◦ Hemolysin
 Four antigenically distinct hemolysins causes hemolysis of erythrocytes.
They are Alpha, Beta, delta and Epsilon toxin, which produce partial
hemolysis (hot – cold hemolysis). The alpha toxin is the major toxin in
gangrenous mastitis. It causes spasm of smooth muscle and is
necrotizing and potentially lethal.
◦ Leukotoxin
 It has the leukocidal activity and includes a and d toxins. By doing so,
the organisms may spread more easily to other parts where they develop
secondary lesions.
◦ Enterotoxin
 It is seldom produced by animal strains. There are several antigenically
distinct types of heat stable enterotoxins. They are not destroyed at 100°C
for 80 minutes. They are responsible for food poisoning in man.
◦ Exfoliative or Dermonecrotoxin
 This toxin causes necrosis of skin by exfoliation and intraepidermal
seperation.
◦ Toxic shock syndrome toxin (TSST)
 Induce excessive lymphokine production and results in tissue damage.
Bovine and human strains of Sta. aureus produce TSST.
Extracellular enzymes

◦ Coagulase
 On their ability to coagulate plasma they are classified as coagulase positive staph.
(CPS) and coagulase negative staph. CPS are considered to be significant pathogens.
They are resistant to heat. The coagulation of plasma produces a fibrin film on the
surface of the organisms, which allows multiplying.
◦ Hyaluronidase
 It hydrolyses hyaluronic acid, the mucoid ground substance of connective tissue.
◦ Nucleases
 Deoxyribonucleases (DNAase) hydrolyze DNA and Ribonucleases hydrolyze RNA.
◦ Fibrinolysin
 Fibrinolysin is commonly referred to as staphylokinase, is an activator of the plasma
system leading to the breakdown of fibrin.
◦ Lipases and esterases - They hydrolyze lipids.
◦ Lysozyme - Hydrolyses the peptidoglycan in the cell wall of many bacteria
 Toxins (Super Antigens)

 Toxic-shock-syndrome toxin
◦ Causes toxic shock syndrome
 Enterotoxins

◦ Stimulate the intestinal muscle contractions, nausea, and

intense vomiting associated with staphylococcal food
poisoning
 Structural Defenses Against Phagocytosis

 S.aureus synthesizes loosely organized polysaccharide slime layers

– Inhibit chemotaxis of and phagocytosis by leukocytes
– Facilitates attachment of Staphylococcus to artificial surfaces
– Protein A coats the cell surface
 Interferes with humoral immune responses by binding Fc region of IgG
antibodies
 Inhibits the complement cascade (part of immune response which pops the
bacterial cell membrane)
Produces Coagulase which converts the soluble blood protein fibrinogen in
insoluble fibrin molecules that form blood clots
Fibrin clots hide the bacteria from phagocytic cells.
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 Staphylococcal Diseases
 Horse: Botryomycosis: Infrequent chronic granulomatous lesions involving the
udder of the mare, cow and sow and the spermatic cord of horses.
 Cattle: Mastitis: Staphylococcal bovine mastitis may be chronic, acute and per-
acute. Gangrenous mastitis due to a toxin is seen in postparturient cows.
 Sheep: Tick pyemia in lambs occurs in 2-5 week old lambs, which is heavily
infected with Ixodes ricinus.
 Poultry

◦ Bumble foot: A pyogranulomatous process of subcutaneous tissue of foot that

can involve the joints.
 Pig: Exudative epidermitis (greasy pig disease) is an acute generalized
infection of suckling and weaned pigs caused by S. hyicus. This disease is
characterized by excess sebaceous secretion, exfoliation and exudation.
Staphylococcus infections

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 Diagnosis
 Cultural characters in selective media.
 In case of food poisoning identification of the enterotoxin is of diagnostic
use. These organisms produced green or greenish brown colonies with white
centres after the milk samples are incubated for 16-20 hours.
 Coagulase test: When culture added to Rabbit plasma, fibrinogen is
converted to fibrin by coagulase enzymes.
 In this test, a suspension of staphylococci is mixed with rabbit plasma either
on a slide or in a small tube.
 A positive reaction is indicated by clumping of bacteria within 1 to2 min.
 It is the definitive test for coagulase production and positive reaction is
indicated by clot formation in the tube following incubation at 37ºC for 24
hrs.
 Molecular: PCR based 16S rRNA Sequencing
Treatment

 Penicillin& Methicilines are the drugs of choice if strains are susceptible.

 New synthetic penicillins such as methicillin, oxacillins are effective.
 Tetracyclines, bacitracin, nitrofurans, Trimethoprims, Cephalosporins,
enrofloxacins are effective.
multidrug-resistant Staphylococcus aureus (MRSA)

 Many MRSA infections occur in hospitals and healthcare facilities,

with a higher incidence rate in nursing homes or long-term care
facilities.
 When infections occur in this manner it is known as healthcare
acquired MRSA or HA-MRSA.
 More serious MRSA infections, especially HA-MRSA infections, are
becoming increasingly difficult to treat.

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Streptococcus

 Gram-positive cocci, arranged in pairs or chains, that are

facultative anaerobes
 Often categorized based on the Lancefield classification

◦ Divides the streptococci into serotype groups based on the

bacteria’s antigens
◦ Lancefield groups A and B include the significant streptococcal
pathogens of humans
 Strep Classification
Genus Streptococcus
Species S. agalactiae, S.dysgalactiae, S. equi subsp
zooepidemicus, S.uberis,
S. equi subsp equi, S. canis, S.suis, S.
pyogenes(human)

Identification

S. pyogenes S. agalactiae S. pneumoniae E. faecalis

Catalase - - - -
Hemolysis beta beta Alpha Alpha or
gamma
Bacitracin sensitive resistant resistant resistant
Bile esculin - - - +
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Pathogenicity

 Structural components
◦ Protein M, which interferes with opsonization and lysis of the
bacteria and a hyaluronic acid capsule, which acts to
camouflage the bacteria
 Enzymes

◦ Streptokinases, deoxynucleases, and C5a peptidase all facilitate

the spread of streptococci through tissues
 Pyrogenic toxins that stimulate macrophages and helper T cells to
release cytokines
 Streptolysins lyse red blood cells, white blood cells, and platelets
 Streptococcus infections

Cattle : Bovine mastitis

 It is caused by S. agalactiae (group B), S. dysgalactiae (grp C), S.
equi subsp. zooepidemicus (grp. C), S. uberis (grp C, D, E, P, V).
 Mastitis arises from the multiplication of streptococci in the teat sinus and
extends into the ducts.
 It causes parenchymatous mastitis, which is characterized by progressively
chronic condition resulting in fibrosis.
 Peptostreptococcus indolicus is an anaerobic streptococcus, which is
responsible for summer mastitis in cattle in association
with Arcanobacterium pyogenes.
Horse
 S. equi and S. equisimilis are the main causes of strangles in young horses.
 It is characterized by a catarrhal discharge, with inflamation of the nasal
mucous membranes, followed by swelling of pharyngeal LN’s in which
abscesses develop.
 The infection spreads through lymph channels. It also causes metritis and
cervicitis in horses.
 Purpura haemorrhagica, considered to be an immune mediated disease, occur
in horses 1 to 3 weeks after illness.
 Bastard strangles – in which abscesses developed in many organs. It is a very
serious complication.
 Chicken
 S. gallinarum causes typical acute septicemia with peritonitis in chicken.
 Dogs
 S. canis is considered to be the cause of acid milk in puppies and canine
tonsillitis. It is also associated with neonatal septicaemia and toxic shock
syndrome.
 Pigs
 S. suis causes porcine cervical lymphadenitis and also isolated from
pneumonia, septicaemia, arthritis, endocarditis, meningitis and reproductive
tract infections.
 It also causes erosive arthritis in young pigs.
 Diagnosis

 Itinvolves clinical, microscopical and bacteriological examination.

 Clinical examination
 Palpation of the udder and supra-mammary lymph nodes will be helpful in
distinguishing the chronic and insidious form of mastitis produced by S.
agalactiae and S. uberis.
 In contrast S. dysgalactiae and S. zooepidemicus causes sudden onset of acute
inflammation of one quarter only with an acute systemic disturbance followed by
joint infections and lameness.
 Microscopical examination
 When long chains of organisms are detected in milk samples from chronic
mastitis, it is caused by S. agalactiae.
 Based on type of haemolysis on blood agar
 Treatment

◦ Penicillin is very effective

 Prevention & Control

◦ Antibodies against M protein provide long-term protection against future
infection of S. pyogenes, but only if it is the same strain
◦ In streptococcal infections in animals, including mastitis, the most
satisfactory method of control is antibiotic therapy using penicillin
preparations to other substances because streptococci develop resistance to
penicillin comparatively infrequently.