Download 1 INTRODUCTION I Bacterial Morphology and Classification

DVM Bacteriology and Mycology. Dr. J. Lewis
INTRODUCTION
I
A.
Bacterial Morphology and Classification
General Features
- prokaryotic (no nuclear membrane)
- most have single chromosomes (some exceptions include Brucella melitensis, Vibrio
cholerae)
- many have extrachromosomal DNA (plasmids)
- small (cocci - 1 μm, spirochaetes - 0.5 x 20 μm)
- need oil immersion (1000x) microscopy for visualization
B. Cell Shape:
1. Cocci (spherical) i.e. Staphylococcus aureus
2. Rods or bacilli (cylindrical)
a. straight rods (Escherichia coli)
b. curved rods (Campylobacter jejuni)
c. branching (Actinomyces bovis)
3. Spiral-coiled - Borrelia, Brachyspira, and Leptospira spp.
4. Variations
S
The term “cocco-bacillary” refers to rods which appear very short or ovoid
(Pasteurella multocida often appears in this form).
S
The term “pleomorphic” refers to various forms as irregular shapes within a single
species or strain (some Corynebacterium spp. may appear as rods and club shapes on the
same slide).
S
Members of the Mycoplasmataceae are also pleomorphic because they lack a cell
wall (they do possess a cell membrane).
C.
Cell Envelope and Surface Structures:
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DVM Bacteriology and Mycology. Dr. J. Lewis
Cell envelope structure determines the outcome to a standard Gram stain reaction. Bacteria
can be classified into two main groups: a) Gram-positive (blue) b) Gram-negative
(pink/red). These staining characteristics represent an important first step in any diagnostic
procedure.
In addition, the cell wall structures that are responsible for different staining characteristics
also determine: the susceptibility of these two main groups to different antimicrobials and
disinfectants, the immunological impact they have on the host animal.
Some Gram staining variations :
- due to autolytic activity (exoglycosylases) on the cell wall of “old” cultures or
anaerobes exposed to air Gram-positive bacteria can stain pink/red and appear as
Gram-negatives (ie. Bacillus anthracis or Clostridium spp.)
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some organisms are called Gram-variable (Erysipelothrix rhusiopathiae, Moraxella
osloensis) and appear as a mix of Gram-positive and Gram-negative.
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Mycobacterium spp. cell walls contain lipids and wax-like substances (mycolic acids)
that make staining with Gram stain unsatisfactory. This group of bacteria are
traditionally visualized using an acid-fast stain protocol.
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Leptospira and Brachyspira spp. are very slender spirochaetes and do not stain well
using the Gram-stain protocol. These bacteria are typically visualized using a Giemsa
or silver-stain protocol.
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bacterial capsules can be visualized using negative-staining protocol (Nigrosin or
India ink).
II Overview of the Bacterial Envelope
A. Typical “core“envelope is composed of several layers including:
1. Inner cell membrane is a symmetrical phospholipid bilayer that is present in both
Gram-positive and Gram-negative bacteria.
2. Cell wall is the peptidoglycan layer that is present in both Gram-negative and Grampositive bacteria (absent in Mycoplasma and Ureaplasma spp.)
3. Integral proteins (including flagella, fimbriae, enzymes, chaperones, etc.).
additional layers (or variations to “core” layers) occur depending on: the Division
(Gracilicutes - Gram negatives), Genus, species or virulence. These additions can
include: outer membrane, S-layer, capsule or slime layer or the absence of a cell wall.
B. Division II: Firmicutes
-
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DVM Bacteriology and Mycology. Dr. J. Lewis
C. Division I.: Gracilicutes
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DVM Bacteriology and Mycology. Dr. J. Lewis
D. Outer Membrane (OM)
1. “Classical” OM occurs only in Gram negative bacteria (see Figure 2)
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OM is an asymmetrical lipid envelope comprised of an inner phospholipid and outer
lipopolysaccharide (LPS) leaflet.
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LPS is comprised of a hydrophobic domain termed Lipid A (or endotoxin), a core
oligosaccharide and a repeating distal polysaccharide (O-antigen).
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LPS functions as a selective permeability barrier, is an extremely important virulence
factor (Lipid A) as well as a serological marker (O-antigen).
E. Periplasmic Space (Gram negatives) - occurs between the inner and outer membranes
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DVM Bacteriology and Mycology. Dr. J. Lewis
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Contains cell wall (peptidoglycan meshwork), soluble proteins (enzymes, chaperones)
Porins (transport channels in the O.M.) are considered to be “Gram-negative” unique
F. O.M. in Gram Positives?
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Recent evidence has postulated a “Gram negative-like” OM in a specific subset of
historically “Gram-positive” organisms (Corynebacterium-Nocardia-Mycobacterium)
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the OM has been suggested to be asymmetrical with a unique lipid called Mycolic acid
occurring in the outer leaflet of the hypothesized membrane.
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these hypothesized OM have been shown to contain porin homologues
G. Variations to the Envelope
1. Glycocalyx:
a
Capsule
-
K-antigen
composed of complex polysaccharides (usually) or polypeptides that
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DVM Bacteriology and Mycology. Dr. J. Lewis
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form a define hydrophilic “gel” that is closely adherent to the outer
surface of many bacteria
- frequently related to virulence (antiphagocytic, adherence)
can be used as source of antigen for vaccination against certain
pathogens (Streptococcus pneumoniae).
b. Slime layer - looser meshwork of polymeric fibrils
2. Flagella -
H-Antigen
-
-protein (flagellin)
responsible for motility
3. Adhesins (Pili and NonPili)
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adhesion (attachment) of bacteria to host cells (frequently mucosal epithelial cells) is
frequently facilitated by “typical” filamentous types of adhesins called pili (also
called fimbriae) or by nonpilus types of adhesins. The “typical” filamentous pili are
deemed virulence factors and made up the protein pilin.
Examples of Pili (Fimbriae) Type Adhesins
a. Thick Rigid Pili
- P-Pili - Uropathogenic E. coli
- Type 1 - Enteropathogenic E. coli
- Type 2 and 3 - Bordetella pertussis
- Type 4 - adhesion, also responsible for “twitch” motility and is a bacteriophage
receptor (Pseudomonas aeruginosa, Moraxella bovis, E. coli, Dichelobacter
nodosus etc.)
- Type 5
b. Thin Flexible Pili
- *K99 - E. coli neonatal diarrhea calves, lambs, pigs
-K88 - E. coli neonatal diarrhea pigs
* Note that “K”-antigen designation was used prior to the new “F-antigen” designation .
4. S-Layer (Rigid Crystalline-Lattice Layer)
- External to cell wall and outer membrane
- Protein or glycoprotein composition
- May facilitate: adhesion, antiphagocytosis, bacteriophage binding.
- Examples of organisms in which an S-layer has been identified (Clostridium
tetani, Campylobacter fetus subsp. fetus)
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DVM Bacteriology and Mycology. Dr. J. Lewis
5. Spores
- Highly resistant, thick-walled oval or spherical bodies, that are produced under
adverse environmental conditions or during later stages of growth. Members of
the genera Clostridium spp. and Bacillus spp. produce spores. The location of the
spore within the parent cell may with identification.
III. Terminology
1. Obligate - absolute requirement for oxygen (aerobes) or the absence of oxygen (obligate
anaerobes).
2. Aerophilic - air loving.
3. Microaerophilic- preference for less than atmospheric levels of oxygen (6%) to grow
(some Actinomyces and Campylobacter spp.).
4. Anaerobic- ie. Bacteroides, Clostridium and Fusobacterium, Brachyspira etc. are strict
(obligate) anaerobes.
5. Facultative - can grow in the presence or absence of oxygen (better in air).
6. Facultative intracellular - may propagate outside or inside host cells.
7. Obligate intracellular - may only propagate when inside host cells.
8. Endospore - spores produced by Clostridium and Bacillus sp. When deprived of
nutrients or growth factors.
9. Genus, species.
10. Subspecies - species subdivision based on small but consistent differences.
11. Strain - is a descendant (clone) of a single isolate. The original pure culture isolate from
which the strain was described is generally deemed the Type Strain.
12. Biovar - Strain with special/unique biochemical or physiological properties.
13. Serovar - Strain with distinctive antigenic properties.
BACTERIAL IDENTIFICATION
I. BACTERIAL CLASSIFICATION
A. General Identification Steps in Diagnostic Unit.
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DVM Bacteriology and Mycology. Dr. J. Lewis
Isolation and Identification of Bacteria from
Clinical Specimens
Specimen
Primary plate media
Blood and MacConkey
24 hr, 37 o C
Direct Smear
and
Stain
Discrete Colonies
Reincubation
Antimicrobial
Sensitivities
37 oC, 24 hr
Pure Culture
Biochemical
Tests
Read
Identification
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Report
Bacteria
from
clinical
specimens
are
identified
to the
DVM Bacteriology and Mycology. Dr. J. Lewis
genus level frequently through the use of simple tests such as: staining characteristics
(Gram stain, acid fast, etc. ), colony and microscopic characteristics, and growth on
selective media.
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Despite the increasing use of molecular technology as diagnostic aids many bacteria are
still identified to the species level on the basis of their reactions to a series of biochemical
tests. However, reclassification of bacteria and fungi is occurring with greater frequency
and relies heavily on DNA sequence data from ribosomal DNA as well as other targets.
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Some pathogens, such as the Salmonella, Leptospira and Escherichia coli may be further
subdivided into serotypes or serovars on the basis of their individual antigenic
repertoires.
II GENETIC CHANGE
A. Mutation: spontaneous or induced changes in genetic character (frequently manifested as
changes in virulence).
B. Horizontal (Lateral) Gene Transfer (Dreiseikelmann, B., 1994, Microbiol. Rev., 58(3):293316.; Cheetham and Katz, 1995, Mol. Micro., 18(2):201-208)
a. Transformation: (Natural and Experimental)
Bacterial cells can acquire naked DNA (plasmid or genomic) from their immediate
environment through specific receptor mediated uptake and translocation mechanisms.
This can occur in both Gram -ve (examples include Moraxella, Neisseria and
Histophilus) and Gram +ve (examples include Bacillus subtilis and Streptococcus
pneumoniae) bacteria.
- virulence-associated genetic information can be acquired in this manner
b. Transduction:
Bacteriophage mediated introduction of novel genetic information into the bacterial
chromosome or plasmids. Phage derived DNA has been demonstrated to introduce a wide
variety of virulence genes involved in the direct or indirect generation of such factors as:
antimicrobial resistance, toxins, LPS and capsules.
c. Conjugation:
Conjugative plasmid is transferred from a donor to a recipient across the envelope of both
bacteria.
Occurs in both Gram -ve and Gram+ve bacteria.
Can occur between closely related species, different genera, between Gram-ve and
Gram+ve, between bacteria and yeast, and between bacteria and plant cells
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DVM Bacteriology and Mycology. Dr. J. Lewis
(Agrobacterium tumefaciens).
Conjugative plasmids tend to be less 30 Kb in size and encode proteins required for
translocation of DNA as well as additional genes encoding antibiotic resistance proteins
and toxins (ie. Enterobacteriacea).
The broad host range potential of conjugative plasmids facilitates antibiotic resistance
transfer between gastrointestinal commensals and pathogenic organisms.
d. Transposition:
Transposons are mobile genetic elements that are generally restricted to movement within
a single host cell and typically integrated in the host chromosome.
However, movement of transposons to plasmids, and conjugative transposons can lead to
movement of transposon encoded genes (such as antibiotic resistance) from one bacterium
to another.
This mechanism can also lead to the accumulation of multi-drug resistance.
Types of Mobile Genetic Elements
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Insertion sequences (IS) - simplest
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Transposon - IS with an Abx resistance gene
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Composite Transposon - two IS flanking genes encoding a number of virulence
factors
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Conjugative Transposon - can mobilize to another bacteria like a conjugative
plasmid but cannot replicate independently.
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DVM Bacteriology and Mycology. Dr. J. Lewis
Pathogen-Host Interactions
Clinical Importance of Understanding Host-Pathogen Relationships:
By utilizing a medical strategy “limited” to diagnosing and treatment (therapeutic as opposed to
prophylactic) there is NO possibility of large scale control or eradication of infectious disease.
Furthermore, by the time a diagnosis is made, the infecting organism has established a foothold in
the host body, and has already caused some damage. At this point, antibiotic treatment “may”
eliminate all of the bacteria from the infected area, but this may not restore the affected animal’s
full health if irreversible tissue damage has been done.
Infection occurs as a results of a complex interaction between the host, the pathogen and the
environment. Patterns of bacterial infections can be related to changes in host (immunity, genetic
make-up), bacteria (virulence, antibiotics) or the host and/or bacterial micro-environment
(transportation, husbandry changes).
I. Definitions and Concepts
Pathogen: An organism that can cause disease.
2. Obligate pathogen: An organism that almost always causes disease when it encounters
animals or humans ( ie. Bacillus anthracis).
3. Primary Pathogen: An organism that generally causes disease when it contacts a susceptible
host.
4. Opportunistic pathogen: These are commensals which can cause disease when they gain
access to other sites or tissues (non-enterotoxin producing E. coli when they gain access to the
urinary tract). Opportunistic infections can arise when the host immune defenses are impaired
(immunosuppression, stress etc.)
Fusobacterium necrophorum (normal resident of G.I. tract in ruminants can cause
liver abscessation when they gain access to the liver).
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DVM Bacteriology and Mycology. Dr. J. Lewis
Intracellular Bacteria
a. Facultative Intracellular organisms described microorganisms that are not confined to the
host intracellular space, however, they can survive and often multiply in the host
cytoplasm. Listeria, Brucella, Mycobacteria, Salmonella all demonstrate this ability.
b. Obligate intracellular organisms must gain access to the host cell intracellular space to
survive and multiply (ie. Chlamydiales and Rickettsiales).
6. Virulence: The degree of pathogenicity of a microorganism as indicated by its ability to
invade the tissues of the host and cause disease. Bacterial virulence is generally multifactorial.
7. Attenuation: The process of diminishing the virulence of a microorganism, often deliberately
in an effort to create an immunologically appropriate vaccine.
8. Infection: The presence of potentially pathogenic organisms established in a host. Note that
presence does not necessarily imply clinical disease (ie. carrier states).
II. Mechanisms of Host Resistance to Disease.
A. Natural or Innate Resistance
1. This type of immunity is rapid and involves a variety of host cell receptors that recognize
unique components of bacterial cells and culminates in the activation and recruitment of
neutrophils and activated macrophages to the sites of infection. This component of
immunity ultimately influences the acquired immune response.
a. The number of host receptors and pathogen ligands that mediate an innate immune
response are well outside the scope of a single lecture. However some key host
receptors include certain Acute Phase Proteins as well as host cell-associated receptors
that include macrophage scavenger receptors and the novel Toll-like receptors
(TLR’s).
i. Acute Phase Proteins
- Complement C3 - binds bacterial CHO’s
- C-Reactive protein and Serum amyloid protein - bind bacterial surfaces and fix
complement
- LPS binding protein (LBP)
- Mannan-binding protein (MBL) - binds exposed terminal mannose residues on
bacteria and can mediate activation of the complement pathway
ii. Macrophage scavenger receptors can bind peptidoglycan (PGN), lipopolysaccharide
(LPS) and lipotechoic acid (LTA)
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DVM Bacteriology and Mycology. Dr. J. Lewis
iii. Toll-like receptors (TLRs) represent a group of recently discovered pattern
recognition receptors that bind a variety of pathogen-associated molecular patterns
(PAMPs). Ten TLRs have been described too date and evidence points to the pivotal
role these ligands play in linking innate to adaptive immunity.
Toll-like receptor
1
2
monocytes
PGN, LTA, LPS (Leptospira), Lipoprotein,
3
LAM
TLR4
monocytes
4
TLR5
monocytes,
NK cells, Tcells
flagella
TLR6
B cells,
monocytes,
NK cells
Mycoplasma lipopeptides
TLR2
Cell Type
Bacterial Ligand(s)
LPS and LTA
TLR9
Dendritic cell 5CpG motifs (ISS’s)
1. In humans
2. In concert with TLR6 and TLR1
3. LipoArabinoMannan from Mycobacterial cell walls
4. TLR-4 actually binds a complex of LPS and LBP (LPS binding protein) and CD14
5, CpG Motifs- (Immune Stimulating Sequences) - The “THIRD GENETIC
CODE”.
- The frequency of occurrence of these dinucleotide pairs is lower in eukaryotes
and plants (termed “CpG suppression”) than in prokaryotes. Also the cytosine
of this dinucleotide pair is methylated in eukaryotes and hypomethylated in
bacteria. These features are responsible for a potent activation of cell-mediated
immune responses in host animals.
2. Host defense peptides - Cationic Antimicrobial Peptides.
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These small peptides may play as important a role as the “conventional” innate and the
acquired immune responses.
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Identified in virtually all species ranging from molluscs to humans and over 500 have
been characterized or identified.
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As a group these peptides demonstrate a very broad spectrum of activity that includes:
Gram-positives, Gram-negatives, fungi, parasites, cancer cells and some enveloped
viruses.
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DVM Bacteriology and Mycology. Dr. J. Lewis
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Tend to be found on parts of the body that would come into contact with
environmental pathogens: ears, eyes, skin and on mucosal epithelial surfaces. Also
found in bone marrow and the testes and comprise the majority of the contents of the
azurophilic granules of PMN’s.
B. Acquired Immunity
1. Passive Immunity
Placental or colostral transfer of pathogen specific antibodies to young
animals. These passive antibodies have a defined serum half-life and offer short term
protection against disease. One drawback of passively acquired antibodies is that they can
interfere in the development of active immunity following vaccination.
2. Active Immunity
Humoral (Antibodies)- bacterins, extracts, subunit vaccines typically induce this arm of
the immune response.
Cell Mediated - often required for clearance of intracellular pathogens (Listeria
monocytogenes). Components of the cell-mediated immune response include T-cells
(Helpers and Cytolytic) and T-cell activated phagocytic cells.
III. Bacterial Interaction with Host (Virulence factors)
A. Attachment/Tissue Invasion factors - These factors allow attachment (leading to
colonization) to the host cells/tissues and include: capsular material, components of the outer
membrane or inner membrane (Mycoplasma adhesins) and pili/fimbriae. Hyaluronidase
(many bacteria) and collagenase (some strains of Clostridium perfringens) are enzymes that
break down host intercellular materials and facilitate the spread of extracellular pathogens.
B. Immune Escape
1. Bacteria, like viruses, utilize a variety of mechanisms to escape the immune surveillance
system of the host. These mechanisms include: antiphagocytic capsules, superantigens,
cytotoxins, coagulase (clots host cell fibrin to impede the movement of host immune
surveillance cells to the bacteria - Staphylococcus aureus)
2. Host cell Invasiveness - Intracellular bacteria typically possess a specific set of virulence
factors necessary to escape the phagolysosome and spread from cell-to-cell.
S Antigenic variation - Bacteria such as Mycoplasma spp. are able to vary the character of
the outer envelope proteins such that antibody recognition is foiled.
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DVM Bacteriology and Mycology. Dr. J. Lewis
S Induced programmed cell death (apoptosis) of host cells involved in manifesting an
immune response. Intact bacteria or certain toxins are able to induce apoptosis and
include: Salmonella, Listeria monocytogenes, Histophilus somni and exotoxin A and
Pseudomonas aeruginosa and Pasteurella haemolytica).
C. Toxins
Exotoxins - a wide variety of bacteria produce exotoxins. The genes encoding these proteins
may be chromosomal, plasmid (tetanus neurotoxin, E. coli enterotoxin, Staphylococcus
enterotoxin) or bacteriophage (diptheria toxin and erythrogenic toxins of Streptococcus
pyogenes) enncoded.
Endotoxins - Lipid A component of lipopolysaccharides (LPS) of Gram -ve bacterial cell
envelopes
D. Biofilms and Quorum Sensing
1. Biofilms represent a “sessile” phase of existence that differs dramatically from the more
conventional “planktonic” or free-living phase.
2. Bacterial biofilms are: a structured community of bacterial cells enclosed in a selfproduced polymeric matrix and adherent to an inert or living surface. This matrix can be
comprised of more than one species and has been shown to contain channels through
which nutrients can circulate in a manner somewhat akin to the tissues of higher
organisms
3. Biofilms are inherently resistant to antimicrobial agents and can give rise to “planktonic”
individuals. Thus they are commonly associated with persistent infections such as dental
caries and periodontitis. They can also play a role in otitis media, necrotizing fasciitis, and
nosocomial infections involving medical devices such as sutures, catheters, IUDs and
heart valves.
4. A variety of common, significant bacterial pathogens can demonstrate a biofilm-phase
including: Staphylococcus aureus and epidermidis, Pseudomonas aeruginosa, Salmonella
spp., Haemophilus influenzae, Escherichia coli, Listeria monocytogenes and more.
5. Quorum Sensing describes cell-to-cell communication between bacteria of the same
species.
- Pseudomonas aeruginosa utilizes a signaling molecule called Acyl-homoserine
lactone to upregulate virulence factors and shift to biofilm formation.
- Staphylococcus aureus uses a peptide pheromone to signal a global upregulation of
virulence factors.
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DVM Bacteriology and Mycology. Dr. J. Lewis
IV. Overview of Bacterial Access/Transmission/Specificity/Control
A. How do bacteria gain access to the body
1. Inhaled, Ingested
2. Through skin or mucosa
3. Via urogenital tract
4. Placenta to fetus
5. Via umbilicus in newborn
B. How does animal transmission occur from animal to animal?
1. Horizontal Transmission
a) Direct
- aerosol
- biting
- venereal
- skin disease by direct contact
b) Indirect (fomites)
- food, water
- bedding, kennels, pens etc.
2. Vertical of Infectious Diseases
- mother to offspring
C. Species and Organ Specificity of Infections
Most of the pathogenic bacteria have a broad host range. However, some are relatively species
specific (ie. Streptococcus equi- horses, S. suis - swine, S. canis , S. agalactiae - cows).
Some bacteria tend to be organ specific, or they have a predilection for certain organs (ie. S.
agalactiae - mammary gland, Brucella abortus - genital organs).
D. Control of Infectious Diseases
1) Source of organisms.
2) Numbers of organisms.
3) Contact
4) Susceptibility of host (Genetics, Vaccination)
Disease = Number of organisms x virulence of organisms
Host Resistance
E. General Steps for Disease Control:
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DVM Bacteriology and Mycology. Dr. J. Lewis
1. Control based on the knowledge of source and transmission of infection
- isolation of diseased animals, carriers and new animals
- environmental eradication and control (infected bedding, water, boots etc.)
- burn or incinerate dead animals
2. Control by decreasing numbers of infectious agents.
- treatment with antimicrobial agents
- good hygienic management practices
3. By decreasing frequency of contacts.
- closed herds or disease free herds should not admit animals within unknown history
- instituting quarantine procedures
- fencing
4. By increasing resistance.
- increase nonspecific resistance (reduction of stress, adequate nutrition, sufficient
ventilation), probiotic therapy.
- Increase specific resistance (vaccination, breeding programs)
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