PHA6114

PHARMACEUTICAL MICROBIOLOGY AND

PARASITOLOGY

BACTERIA
ASST. PROF. RHONA P. RAMOS, MSc.
Course Facilitator
 bacteria
plants
animals

archaea
fungi
protists
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diverse /dəˈvərs, dīˈvərs/ adj.
showing a great deal of variety; very
different

UBIQUITOUS /yo͞ oˈbikwədəs/ adj.

present, appearing, or found
everywhere
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ULTRASTRUCTURE
1–5 μm

smallest free-living organisms

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ULTRASTRUCTURE

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 COCCUS DIPLOCOCCI

STAPHYLOCOCCI STREPTOCOCCI
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Neiserria gonorrhea Staphylococcus aureus Streptococcus pyogenes Sarcina ventriculli

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Klebsiella pneumoniae Bacillus cereus Bordetella pertussis Corynebacterium diphtheria

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Vibrio cholera Treponema pallidum

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 ULTRASTRUCTURE

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 ULTRASTRUCTURE

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 ULTRASTRUCTURE
cell wall
maintenance of the shape and integrity of the bacterial cell
withstand the osmotic pressures
obvious target for antibiotics

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 PEPTIDOGLYCAN
ULTRASTRUCTURE

large macromolecule containing glycan

(polysaccharide) chains that are cross-
linked by short peptide bridges

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 L-alanine
D-glutamic acid
lysine or
diaminopimelic acid
(DAP)
D-alanine,

N-acetyl muramic acid (NAM)

N-acetyl glucosamine (NAG)

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Gram stain

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LPS

it determines the antigenicity of the Gram-negative cell

and it is extremely toxic to animal cells

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LPS

lipid A component is responsible for the

toxic and pyrogenic properties of Gram-
negative bacteria

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OUTER MEMBRANE
not permeable to enzymes or large molecules
not readily penetrated by hydrophobic compounds and
is, therefore, resistant to dissolution of detergents

PERIPLASM
in between cytoplasmic
membrane and outer
membrane

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 ULTRASTRUCTURE

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 ULTRASTRUCTURE
CYTOPLASMIC MEMBRANE

fragile, phospholipid bilayer with proteins distributed

randomly throughout
prokaryotes do not have sterols in their membranes (e.g.
cholesterol)

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 ULTRASTRUCTURE
CYTOPLASMIC MEMBRANE

transport of nutrients, energy generation and electron

transport
location for regulatory proteins and biosynthetic proteins
semipermeable selectivity barrier between the cytoplasm
and the cell environment

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 ULTRASTRUCTURE
MESOSOMES

invaginations
organs of attachment for the bacterial chromosome

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 CYTOPLASM

contains enzymes that generate ATP

directly by oxidizing glucose and other
carbon sources
contains some of the enzymes involved in
the synthesis of peptidoglycan subunits

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NUCLEOID

singular, covalently closed circular molecule of double-

stranded DNA comprising approximately 4600 kilobase pairs
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PLASMIDS

small, circular pieces of double-

stranded extrachromosomal DNA
capable of autonomous replication

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PLASMIDS

antibiotic resistance
transfer readily from one organism
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RIBOSOMES

70S in size
made up of two subunits of 30S
and 50S
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INCLUSION GRANULES

consist of storage material composed of carbon, nitrogen,

sulphur or phosphorus
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Cell surface components
Flagella

provides bacterial motility

long (c.12 µm) helical-shaped
structures that project from
the surface of the cell
filament is built up from
multiple copies of the protein
flagellin

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 Cell surface components

Pseudomonas aeruginosa

E. coli

Spirillum volutans
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Cell surface components
FIMBRIAE

structurally similar to flagella, but

are not involved in motility
straighter, more numerous and
considerably thinner and shorter
(3µm)
act as adhesins

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Cell surface components
PILI

similar to fimbriae, but they are present in much smaller

numbers (<10) and are usually longer
genetic exchange process of conjugation rpramos2019
Cell surface components
CAPSULES AND SLIME LAYERS

capsules, which form a tight, fairly rigid layer closely

associated with the cell
slimes, which are loosely associated with the cell rpramos2019
Cell surface components
S-LAYERS

two-dimensional paracrystalline array of

proteins or glycoproteins
present on the outer surfaces in addition
to other cell wall components such as
polysaccharides

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BIOFILMS

multilayered communities of usually more than one species

of bacteria as a functional, dynamic consortium
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BIOFILMS

EPS hold the bacterial cells at a distance from one another to

form small water channels
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BIOFILMS

the resident cells within the biofilm are not exposed to attack by
the immune system and in some instances can exacerbate the
inflammatory response rpramos2019
BIOFILMS

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BIOFILMS

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BIOFILMS

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SPORULATION

Bacillus anthracis Clostridium difficile

vegetative cell undergoes a profound biochemical change to

give rise to a specialized structure called an endospore or spore
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SPORULATION
ENDOSPORE STRUCTURE

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SPORULATION
ENDOSPORE FORMATION

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SPORULATION
ENDOSPORE GERMINATION

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 pathogens - damage host in different ways

OPPORTUNIST PATHOGENS

organisms that, if presented with the correct set of conditions, can cause
disease
Staphylococcus epidermidis, a beneficial organism when present on the
skin yet potentially fatal if attached to a synthetic heart valve
Ps. aeruginosa, a non-pathogenic environmental organism but again
potentially lethal in immunocompromised patients

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TOXINS
products of bacteria that produce immediate host cell damage
endotoxin, i.e. cell wall-related
exotoxin, products released extracellularly as the organism grows

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TOXINS
ENDOTOXIN

lipid A component of LPS

ability to induce fever, initiate the complement and blood cascades, activate
B lymphocytes and stimulate production of tumour necrosis factor
released from lysed or damaged cells
heat-resistant material
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TOXINS
EXOTOXIN

1. A-B toxins
consist of a B subunit that binds to a host cell receptor and is also
covalently bound to the A subunit that mediates the enzymic
activity responsible for toxicity
diphtheria toxin, cholera toxin

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TOXINS
EXOTOXIN

2. cytolytic toxins
work by enzymatically attacking cell constituents, causing lysis
haemolysins and phospholipases rpramos2019
TOXINS
EXOTOXIN

3. superantigen toxins
act by stimulating large numbers of immune response cells to
release cytokines, resulting in a massive inflammatory reaction
Staphylococcus aureus-mediated toxic shock syndrome

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REPRODUCTION AND GROWTH KINETICS
BINARY FISSION

each individual will increase in size until it is large enough to divide into two
identical daughter cells
each must carry at least one copy of the chromosome
the time taken to copy an entire chromosome depends on the number of
base pairs within it and the growth temperature
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REPRODUCTION
BINARY FISSION

45 min
15 min

rods elongate gram (-) constriction

cocci expands radially gram (+) cross wall formation
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GROWTH KINETICS
generation time

time interval between one cell division and the next

number of bacteria n in any generation can be expressed
as: xth generation n = 1 x 2x

N = No × 2x

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GROWTH KINETICS

n = 1 x 21 n = 1 x 22 n = 1 x 23
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GROWTH KINETICS

colonies
on solid surface, with nutrients and moisture
easily visible to the naked eye

agar media
nutrient soup (broth) that has been
solidified by the addition of agar
Petri dish or plate, or bottle (slant)

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GROWTH KINETICS
CLOSED SYSTEM

waste products of metabolism are retained and all the available

nutrients are present at the beginning of growth

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GROWTH KINETICS
OPEN SYSTEM

continual supply of fresh nutrients and removal of waste products

bacteria in our gastrointestinal tracts receive a more or less continuous
supply of food and excess bacteria are voided with the faeces

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GROWTH KINETICS
GENETIC EXCHANGE - TRANSFORMATION
ability of certain types of bacteria to absorb small pieces of naked DNA
from the environment that may recombine into the recipient chromosome
transferring genes between different types of bacteria

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GROWTH KINETICS
GENETIC EXCHANGE -
TRANSDUCTION
a process of gene
movement by
bacteriophages

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GROWTH KINETICS
GENETIC EXCHANGE - CONJUGATION

transfer process where unassociated

F-factor will simply transfer a copy
of DNA to a recipient cell

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GROWTH KINETICS
GENETIC EXCHANGE

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FACTORS FOR GROWTH AND SURVIVAL
TEMPERATURE

permissive temperatures -
range of temperatures
under which bacteria can
actively grow and multiply

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FACTORS FOR GROWTH AND SURVIVAL
pH

pH effects on growth are bell-shaped

microorganisms that have medical or
pharmaceutical significance have pH
growth optima of between 7.4 and 7.6 but
may grow suboptimally at pH values of 5–
8.5

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FACTORS FOR GROWTH AND SURVIVAL
WATER ACTIVITY (AW)
vapour pressure of water in the space above the material relative to the
vapour pressure above pure water (1.00) at the same temperature and
pressure
pharmaceutical creams might have Aw values of 0.8–0.98
Gram-negative bacteria cannot grow if the Aw is below 0.97, whereas Gram-
positive bacteria can grow in materials with Aw of 0.8–0.98
can markedly affect the vulnerability of pharm’l products to spoilage
contaminants

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FACTORS FOR GROWTH AND SURVIVAL
OXYGEN

aerobic microorganisms - oxygen acts as the terminal electron acceptor in

respiration and is essential for growth
obligate anaerobes - bacteria for which oxygen is highly toxic
close proximity of strongly aerobic cells and anaerobes will create an anoxic
microenvironment in which the anaerobe can flourish

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FACTORS FOR GROWTH AND SURVIVAL
NUTRITION
carbon, nitrogen, water, phosphorus, potassium and sulphur with a minor
requirement for trace elements such as magnesium, calcium, iron, etc.
chemolithotrophs - utilize atmospheric carbon dioxide and nitrogen as sources
of carbon and nitrogen

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FACTORS FOR GROWTH AND SURVIVAL
NUTRITION
auxotrophs - can grow on simple sugars together with ammonium ions, a
source of potassium and trace elements
pseudomonads and Achromobacter species

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 FACTORS FOR GROWTH AND SURVIVAL
NUTRITION

diauxic growth - growth in liquid cultures

with dual provision of substrate
characterized by a second lag phase
during the logarithmic growth period
while adaptation takes place

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detection

CULTURE TECHNIQUES
to assess the total numbers of specific groups of microorganisms or to
determine the presence or absence of particular named species

ENUMERATION

to dilute the sample to varying degrees and inoculate the surface of a

predried nutrient agar with known volumes of those dilutions
will produce visible colonies that can be counted and the numbers
related back to the original sample
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detection
ENUMERATION MEDIA
will only ever culture a subset of cells towards which the medium and
incubation conditions are directed
temperatures akin to the human body are often deployed because only
those bacteria able to grow at such temperatures are likely to cause
infection
highly nutritious media, e.g. blood agar

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detection
ENUMERATION MEDIA

settle plates - agar plates deliberately exposed to air

number of colonies formed is related to the bacteria content of a room
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detection
RAPID ENUMERATION TECHNIQUES
bioluminescence, epifluorescence and impedance techniques
for pharmaceutical waters and aqueous pharmaceutical products

Coulter counters
determine bacterial concentration
microcalorimeters
detect microbial growth through
detection of heat

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detection
ENRICHMENT CULTURE
intended to increase the dominance of a numerically
minor component of a mixed culture
always liquid
MacConkey broth contains bile salts that will inhibit
the growth of non-enteric bacteria and may be
used to enrich for Enterobacteriaceae

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detection
SELECTIVE MEDIA
solidified enrichment broths
intended to suppress the growth of particular
groups of bacteria and to allow the growth of
others
mannitol salts agar will favour the growth
of micrococci and staphylococci
cetrimide agar will favour the growth of
pseudomonads

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detection
IDENTIFICATION MEDIA
contain nutrients and reagents that indicate, usually
through some form of color formation, the presence of
particular organisms
Salmonella spp. Escherichia spp.
Shigella spp. Klebsiella spp.

non-fermenting lactose-fermenting
MacConkey agar
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detection
IDENTIFICATION MEDIA
inclusion of egg-yolk lecithin into an agar gives it a cloudy
appearance that clears around colonies of organisms that produce
lecithinase (a virulence factor in staphylococci)

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MICROSCOPY

simple stains
size and shapes of individual cells
arrangement into clusters, chains and tetrads
specific stains for the presence of endospores,
capsules, flagella and inclusion bodies
motility status

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molecular approach to identification
denaturing gradient gel electrophoresis; DGGE
isolates and amplifies 16S ribosomal DNA and, following
sequencing of the bases, compares this with known sequences
held in a reference library

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 Problematic
Useful Pharmaceutical
Organism Characteristic Pharmaceutical
Relevance
Relevance

Kelsey-Sykes Skin contaminant

Staphylococcus Gram-positive, aerobic, disinfectant capacity Food poisoning
aureus catalase-positive cocci test Toxic shock syndrome
Preservative limit test Pyogenic infections

Implanted medical
Staphylococcus Gram-positive, aerobic, device/prosthetic device
epidermidis catalase-positive cocci contaminant
Biofilm-former

Causative agents of
Gram-positive, aerobic,
Streptococcus spp. tonsilitis and scarlet
catalase-negative cocci
fever

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 Useful Pharmaceutical Problematic
Organism Characteristic
Relevance Pharmaceutical Relevance

Used to validate and

Bacillus atrophaeus
Gram-positive rod, aerobic, monitor dry heat and
(formerly Bacillus
spore-former ethylene oxide
subtilis)
sterilization processes
Used to validate and
Gram-positive rod, aerobic,
Bacillus pumilus monitor radiation
spore-former
sterilization processes
Geobacillus
Used to validate and
stearothermophilus Gram-positive rod, aerobic,
monitor moist heat
(formerly Bacillus spore-former
sterilization processes
stearothermophilus)
Used to confirm
Gram-positive rod, anaerobe,
Clostridium sporogenes anaerobic growth
spore-former
conditions

Gram-positive rod, anaerobe,

Clostridium tetani Vaccine against tetanus Causative agent of tetanus
spore-former
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 Problematic
Useful Pharmaceutical
Organism Characteristic Pharmaceutical
Relevance
Relevance

Leuconostoc
Gram-positive rod Dextran production
mesenteroides

Corynebacterium Gram-positive rod, Vaccine against Causative agent of

diphtheriae aerobe diphtheria diphtheria

Causative agent of
Mycobacterium Gram-positive, acid-fast tuberculosis
tuberculosis rod, aerobe Disinfectant resistance
Intracellular pathogen

Gram-positive,
Actinomyces spp. Antibiotic production
filamentous rods
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 Problematic
Useful Pharmaceutical
Organism Characteristic Pharmaceutical
Relevance
Relevance

Vaccine against Causative agent of

Bordetella pertussis Gram-negative rod, aerobe
whooping cough whooping cough

Causative agent of
Haemophilus
Gram-negative rod, aerobe infantile meningitis and
influenzae
chronic bronchitis

Haemophilus Vaccine against Hib

Gram-negative rod, aerobe
influenzae type b infections

Legionella Causative agent of

Gram-negative rod, aerobe
pneumophila Legionnaire’s disease

Kelsey–Sykes
Proteus vulgaris Gram-negative rod, aerobe
disinfectant capacity test
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 Problematic
Useful Pharmaceutical
Organism Characteristic Pharmaceutical
Relevance
Relevance

Kelsey–Sykes
Gram-negative enteric disinfectant capacity Food poisoning,
Escherichia coli
rod, facultative anaerobe test Preservative limit severe enteritis
test

Chick Martin/Rideal
Salmonella enterica Gram-negative enteric
Walker disinfectant
serovar Typhi rod, facultative anaerobe
coefficient test

Gram-negative enteric Varying degrees of

Salmonella spp. rods, facultative food poisoning,
anaerobes typhoid fever
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 Problematic
Useful Pharmaceutical
Organism Characteristic Pharmaceutical
Relevance
Relevance

Gram-negative enteric spiral

Campylobacter jejuni Severe enteritis
rod, microaerophilic

Brevundimonas
(formerly 0.22 μm filter challenge
Gram-negative, microaerobic
Pseudomonas test
diminuta)

General environmental
contaminant
Alginate production Quintessential
Pseudomonas
Gram-negative, microaerobic Kelsey–Sykes opportunist pathogen
aeruginosa
disinfectant capacity test High resistance to
antibiotics and biocides
Biofilm-former
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 Useful Problematic
Organism Characteristic Pharmaceutical Pharmaceutical
Relevance Relevance

Gram-negative
Bacteroides
enteric rod, Wound infections
fragilis
anaerobe

Neisseria Gram-negative Vaccine against

meningitidis cocci, aerobic meningitis C

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