Frank Lowy

Bacterial Classification, Structure and Function

Introduction
The purpose of this lecture is to introduce you to terminology used in microbiology. The lecture
will:
1. cover different classification schemes for grouping bacteria, especially the use of the Gram
stain
2. describe the different types of bacteria
3. discuss bacterial structure and the function of the different bacterial components
4. discuss the distinguishing characteristics of Gram positive and Gram negative bacteria.

For this lecture you should focus on the major concepts and not on the names of the
different bacteria. They are mentioned as illustrations of different principles. You will see them all
again as the course progresses.

Classification Systems
The classification of bacteria serves a variety of different functions. Because of this variety,
bacteria may be grouped using many different typing schemes. The critical feature for all these
classification systems is that the organism identified by one individual (scientist, clinician,
epidemiologist), is recognized as the same organism by another individual. At present the typing schemes
used by clinicians and clinical microbiologists rely on phenotypic typing schemes. These schemes utilize
the bacterial morphology and staining properties of the organism, as well as O2 growth requirements of
the species combined with a variety of biochemical tests. For clinicians the environmental reservoir of the
organism, the vectors and means of transmission of the pathogen are also of great importance. The
classification schemes most commonly used by clinicians and clinical microbiologists are discussed
below.
Scientists interested in the evolution of microorganisms are more interested in taxonomic
techniques that allow for the comparison of highly conserved genes among different species. As a result
of these comparisons a phylogenetic tree can be developed that displays the degree of relatedness of
different organisms. A relatively new application of this technology has been the recognition and
characterization of noncultivatable pathogens and the diseases that they cause.

Phenotypic classification systems: There is a chart at the end of these lecture notes on the general
phenotypic classification of many of the clinically important bacteria. This is provided as a reference. By
the end of the course you will be able to recognize most of these microorganisms.

Gram stain and bacterial morphology: Of all the different classification systems the
Gram stain has withstood the test of time. Discovered by H.C. Gram in 1884 it remains an
important and useful technique to this day. It allows a large proportion of clinically important
bacteria to be classified as either Gram positive or negative based on their morphology and
differential staining properties. Slides are sequentially stained with crystal violet, iodine, then
destained with alcohol and counter-stained with safranin. Gram positive bacteria stain blue-
purple and Gram negative bacteria stain red. The difference between the two groups is believed
to be due to a much larger peptidoglycan (cell wall) in Gram positives. As a result the iodine and
crystal violet precipitate in the thickened cell wall and are not eluted by alcohol in contrast with
the Gram negatives where the crystal violet is readily eluted from the bacteria. As a result
bacteria can be distinguished based on their morphology and staining properties.

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 Some bacteria such as mycobacteria (the cause of tuberculosis) are not reliably stained due to the
large lipid content of the peptidoglycan. Alternative staining techniques (Kinyoun or acid fast
stain) are therefore used that take advantage of the resistance to destaining after lengthier initial
staining.

Growth Requirements: Microorganisms can be grouped on the basis of their need for oxygen to
grow. Facultatively anaerobic bacteria can grow in high oxygen or low oxygen content and are
among the more versatile bacteria. In contrast, strictly anaerobic bacteria grow only in conditions
where there is minimal or no oxygen present in the environment. Bacteria such as bacteroides
found in the large bowel are examples of anaerobes. Strict aerobes only grow in the presence of
significant quantities of oxygen. Pseudomonas aeruginosa, an opportunistic pathogen, is an
example of a strict aerobe. Microaerophilic bacteria grow under conditions of reduced oxygen
and sometimes also require increased levels of carbon dioxide. Neisseria species (e.g., the cause
of gonorrhea) are examples of micraerophilic bacteria.

Gram Positive Bacteria

Miscellaneous / Poorly Staining Species
Cocci Rods

Aerobic Anaerobic Aerobic Anaerobic Intracellular Bacteria Poorly Staining Acid Fast Stain
Chlamydia Mycoplasma Mycobacteria
Staphylococci Peptostreptococci* Bacillus* Actinomyces Rickettsia Legionella Nocardia* (modified)
Streptococci Listeria Clostridium Borellia Helicobacter
Enterococci Nocardia*

Gram Negative Bacteria

Cocci Rods

Aerobic Anaerobic Aerobic Anaerobic

Facultative Anaerobe Facultative Anaerobe

Neisseria Veillonella* Enterobacteriaceae Pseudomonas Bacteroides

Branhamella* Vibrio Fusobacterium
Hemophilus

Lactose fermenters Nonlactose fermenters

E. coli Salmonella
Klebsiella Shigella

Biochemical reactions: Clinical microbiology laboratories typically will identify a pathogen in

a clinical sample, purify the microorganism by plating a single colony of the microorganism on a
separate plate, and then perform a series of biochemical studies that will identify the bacterial
species.

Serologic systems: Selected antisera can be used to classify different bacterial species. This
may be based on either carbohydrate or protein antigens from the bacterial cell wall or the
capsular polysaccharide. (Group A streptococcal M proteins or O and H polysaccharide antigens
of salmonella).

Environmental Reservoirs: When considering likely pathogens it is also important to know

which of the different species are found in different locations. Environmental reservoirs are
generally divided into those that are endogenous (i.e., on or within the human body) and
exogenous (somewhere in the environment). When considering the likely cause of an infection
the likely source of the infection is important in your differential diagnosis. For example an
abscess that develops after large bowel surgery is likely caused by an anaerobic organism that is
resident in the large bowel. A skin rash developing in a hiker with a history of multiple tick bites
is more likely to be borrelia, the agent of Lyme disease. An outbreak of food poisoning traced to
imported unpasteurized cheese might be due to listeria.

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 Endogenous reservoirs account for a large proportion of human infections. Many parts of the
body have their own normal flora. S. epidermidis is found on the skin. Viridans streptococci are a
part of the normal oropharyngeal flora and S. aureus is a commensal of the anterior nares.

Genotypic systems:

Universal Phylogenetic Tree: Woese has developed a “universal phylogenetic tree” for all
living organisms that establishes a tripartite division of all living organisms– bacteria, archaea
and eucarya. His work is based on a comparison of 16s ribosomal RNA sequences. These
sequences are highly conserved and undergo change at a slow, gradual and consistent rate. They
are therefore useful for making comparisons among the different living organisms.

Ribosomal RNA (rRNA) sequence analysis: This has emerged as a major method for
classification. It has been used (as described above) to establish a phylogenetic tree. In addition it
is now also used to rapidly diagnose the pathogen responsible for an infection, to help select
appropriate therapy and to identify noncultivatable microorganisms.

Molecular subtyping: Sometimes it is necessary to determine whether strains from the same
species are the same or different. For example if there is an outbreak of infections that appear due
to the same bacterial species, the hospital epidemiologist will want to know if all of the infections
are due to the same strain. This may be done by examining the biochemical studies or the
antibiotic susceptibility profile but a more reliable method is by molecular analysis. Pulsed Field
Gel Electrophoresis (PFGE) is the most frequently used molecular technique. Chromosomal
DNA is digested with a restriction enzyme that makes relatively infrequent cuts in the DNA and
as a result creates large DNA fragments. The DNA fragments from the different strains are then
run on a gel and compared.

Prokaryotes – Structure/Function
Prokaryotes are distinguished from eukaryotes by their smaller size (0.2-10µm), their lack of
internal organelles (e.g., mitochondria), the presence of a cell wall and their cell division by binary fission
rather than mitosis. They lack introns, are not capable of endo/exocytosis and have single-stranded
circular DNA rather than multiple discrete chromosomes.

Bacteria share a number of common structures that are briefly described below.

1) Slime (extracellular polysaccharide) This is extracellular material, loosely associated with

the bacteria, that is elaborated by some bacterial species that facilitates colonization of
smooth, prosthetic surfaces such as intravascular catheters.
2) Capsule This polysaccharide outer coating of the bacterial surface often plays a role in
preventing phagocytosis of bacteria.
3) Peptidoglycan (cell wall) Provides bacterial shape and rigidity. The cell wall consists of
alternating units of N-acetylglucosamine and N-acetylmuramic acid. The polysaccharide
chains are cross-linked by a peptide bridge. It is a primary target of antimicrobial therapy –
because it is specific to prokaryotes.

Assembly of the peptidoglycan:

This is a critical step for bacterial survival. The sequence of events is outlined below.
i. Synthesis begins with formation of a water soluble, nucleotide-linked precursor
(N-acetylmuramic acid - NAM) also carrying a pentapeptide in the cytoplasm.
ii. The precursor is then linked to a lipid-like carrier in the cell membrane

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 (bactoprenol) and N-acetyl glucosamine (NAG) is added to the NAM. This
complex is mobilized across the cytoplasm
iii. The disaccharide subunit (NAM-NAG) is then added to the end of a glycan
strand.
iv. The final step is the transpeptidation reaction catalyzed by a transpeptidase
enzyme (also called penicillin binding proteins) that crosslinks the growing
strand with others.

4) Cytoplasmic membrane This is a phospholipid bilayer that assumes many of the functions
of eukaryotic organelles such as the biosynthetic processes.
5) Flagella These provide bacteria with the capacity for locomotion. They vary in number
and location.
6) Pili These structures project from the cell surface enabling bacteria to adhere to host tissue
surfaces. Based on their amino acid structure their affinity for particular host tissue surfaces
can be remarkably specific.
6) Secreted products There are a variety of these products including exotoxins that are
proteins grouped into A-B toxins (such as those elaborated by vibrio, the cause of cholera),
membrane damaging toxins (e.g., hemolysins) and hydrolytic enzymes which are capable of
destroying host tissues and extracellular matrices.

Distinguishing Features between Gram Positive and Negative Bacteria

Gram positive bacteria have a large peptidoglycan structure. As noted above, this accounts for the
differential staining with Gram stain. Some Gram positive bacteria are also capable of forming spores
under stressful environmental conditions such as when there is limited availability of carbon and
nitrogen. Spores therefore allow bacteria to survive exposure to extreme conditions and can lead to re-
infection (e.g., pseudomembranous colitis)

Gram negative bacteria have a small peptidoglycan layer but have an additional membrane, the outer
cytoplasmic membrane. This creates an additional permeability barrier and results in the need for
transport mechanisms across this membrane.

A major component of the cytoplasmic membrane that is unique to Gram negatives is endotoxin. This
component is essential for bacterial survival. Endotoxin has three components: the lipid A moiety, the
highly conserved core polysaccharide and the species specific O antigen (also polysaccharide). In contrast
with the secreted exotoxins, endotoxin is cell associated but with bacterial division and death can be
released. The Lipid A moiety of endotoxin is responsible for sepsis and this may be fatal. It is
characterized clinically by confusion, fever, drop in blood pressure and ultimately multi-organ failure.

References: Murray et al., Chapters 2 - pages 7-10, 3 - pages 11-24 and 9 - pages 83-87.
(For additional information or clarification)

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 General Phenotypic Classification of Bacteria

Gram Positive Bacteria

Reservoirs / Sites
O2 Require- Commens- Types of
Name Morphology of colonization,
ments al Infections
Transmission
Staphylococci Cocci in facultative Yes Skin, nares / Soft tissue,
grape-like anaerobe endogenous, direct bone, joint,
clusters contact, aerosol endocarditis,
food poisoning
Streptococci Cocci in pairs, facultative Some Oropharynx, skin / Skin,
chains anaerobe species endogenous, direct pharyngitis,
contact, aerosol endocarditis,
toxic shock
Pneumococci Diplococci, facultative ± Oropharynx, sinus / Pneumonia,
lancet shaped anaerobe aerosol otitis, sinusitis,
meningitis
Enterococci Cocci in pairs, facultative Yes GI tract / UTI, GI,
chains anaerobe endogenous, direct catheter-related
contact infections
Bacilli Rods, spore- aerobic ± Soil, air, water, Anthrax, food
forming animals / aerosol, poisoning,
contact catheter-related
infections
Clostridia Rods, spore anaerobic Some GI tract, soil / Breach Tetanus,
formers species of skin, endogenous, diarrhea, gas
ingestion gangrene,
botulism
Corynebac- Rods, facultative Some Skin Catheter-related
terium nonspore anaerobe species infections,
forming diphtheria
Listeria Rods, facultative No Animals, food Meningitis
nonspore anaerobe products / Ingestion
formers
Actinomyces Irregular, anaerobic Yes GI tract / Skin, soft tissue
filamentous, endogenous
form sulfur
granules

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 Gram Negative Bacteria
Reservoirs / Sites
O2 Require- Commens- Types of
Name Morphology of colonization,
ments al Infections
Transmission
Enterobact-
eriaceae Diarrhea,
GI tract, animals /
(E. coli, facultative Some urinary tract,
Rods Endogenous, fecal
klebsiella, anaerobe species food poisoning,
oral
salmonella, sepsis
shigella)
Abscesses,
GI tract /
Bacteroides Rods anaerobic Yes intraabdominal
Endogenous
infections
Water, soil / Infections in
Pseudomon Endogenous, immunocompr
Rods aerobic No
as breach of skin omised hosts,
barrier Cystic Fibrosis
Water /
Vibrio Rods, curved microaeroph
No Contaminated Diarrhea
(cholera) shape ilic
food, water
Campylobac Rods, curved microaeroph Food / Ingestion of Diarrhea,
No
ter shape ilic contaminated food Bacteremia
Rods, poorly microaeroph Water / Inhalation Pneumonia,
Legionella No
stained ilic of aerosol febrile illness
No (N. Meningitis,
Cocci,
Microaero- meningitidi Humans / Sexual , pelvic
Neisseria kidney-bean
philic s aerosol inflammatory
shaped
sometimes) disease
Coccobacillar Respiratory tract / Respiratory,
facultative Some
Hemophilus y- Endogenous, sinusitis, otitis
anaerobe species
pleomorphic aerosol meningitis
Cat scratch
Small, aerobic / Cats, fleas, lice / disease,
Bartonella pleomorphic microaero- No cat bites, lice or endocarditis,
rods philic fleas? bacillary
angiomatosis

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 Miscellaneous Bacteria
Reservoirs /
O2 Require- Commen Sites of Types of
Name Morphology
ments sal Colonization, Infections
Transmission
Helicobacter Not visible on microaero- Yes Stomach / peptic ulcer
Gram stain - philic Endogenous, disease, gastric
helical Fecal-oral ulcer
(corkscrew)
shaped
Mycobacteria Rods, Weakly aerobic No Lungs / Tuberculosis
Gram positive, Fomites
Acid fast stain
positive
Treponemes Not visible on nonculturable No Humans / Syphilis
Gram stain, on routine Sexual
spiral shaped media transmission
on dark field
exam
Borrelia Not visible on nonculturable No Rodents, Ticks Lyme, Relapsing
Gram stain, on routine / Tick bites fever
spiral shaped media
on dark field
exam
Mycoplasma Not visible on Non- Some Humans / Respiratory tract
Gram stain, culturable on species aerosol infections
no cell wall, routine
pleomorphic media
Rickettsia/ Obligate Non- No Ticks, Mites/ Cause a variety
Ehrlichia intracellular culturable on transmitted of illnesses
(Gram routine from the feces including
negative but media of infected lice, systemic
not visible on fleas, ticks vasculitis (e.g.
Gram Stain) Rocky Mountain
Spotted Fever),
rash, pneumonia

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