Chapter 2 Bacterial structure and taxonomy

Classification of all living beings, including microbes has been attempted by many over centuries (Table 2.1). Traditionally, though they were all classified into two kingdoms, plants and animals, classification was arbitrary and based on morphological and growth characteristics. With the development of novel techniques, the latter classification was expanded to include five kingdoms: monera, protista, plantae, fungi and animalia. However, the current understanding based on their genetic relatedness is that all forms of life fall into three domains: Archaea, Bacteria and Eucarya. The main differences among Archaea, Bacteria and Eucarya are listed in Table 2.2. Note that taken together, Archaea and Bacteria are also known as prokaryotes (see below).

Table 2.1 Differential characteristics of major groups of organisms

Table 2.2 Major differences among the three domains of life

Bacteria	Archaea	Eucarya
Organization of the genetic material and replication
DNA free in the cytoplasm	DNA free in the cytoplasm	DNA is contained with a membrane-bound nucleus. A nucleolus is also present
Only one chromosome	Only one chromosome	More than one chromosome. Two copies of each chromosome may be present (diploid)
DNA associated with histone-like proteins	DNA associated with histone-like proteins	DNA complexed with histone proteins
May contain extrachromosomal elements called plasmids	Plasmids may be found	Plasmids only found in yeast
Introns not found in mRNA	Introns not found in most genes	Introns found in all genes
Cell division by binary fission – asexual replication only	Reproduce asexually and spores are not found	Cells divide by mitosis
Transfer of genetic information occurs by conjugation, transduction and transformation (see Chapter 3)	Processes similar to bacterial conjugation enables exchange of genetic material	Exchange of genetic information occurs during sexual reproduction. Meiosis leads to the production of haploid cells (gametes), which can fuse
Cellular organization
Cytoplasmic membrane contains hopanoids	Membranes contain isoprenes	Cytoplasmic membrane contains sterols
Lipopolysaccharides and teichoic acids found	No lipopolysaccharides or teichoic acids found
Energy metabolism associated with the cytoplasmic membrane		Mitochondria present in most cases
Photosynthesis associated with membrane systems and vesicles in cytoplasm		Chloroplasts present in algal and plant cells
		Internal membranes, endoplasmic reticulum and Golgi apparatus present associated with protein synthesis and targeting
		Membrane vesicles such as lysosomes and peroxisomes present
		Cytoskeleton of microtubules present
Flagella consist of one protein, flagellin	Contains flagella that derive energy from proton pumps	Flagella have a complex structure with 9 + 2 microtubular arrangement
Ribosomes – 70S	Ribosomes behave more like eucarya when exposed to inhibitors	Ribosomes – 80S (mitochondrial and chloroplast ribosomes are 70S)
Peptidoglycan cell walls	Cell walls lack peptidoglycan	Polysaccharide cell walls, where present, are generally either cellulose or chitin

Viruses are not included in this classification as they are unique, acellular, metabolically inert organisms and therefore replicate only within living cells. Other differences between viruses and cellular organisms include:

• Structure. Cells possess a nucleus or, in the case of bacteria, a nucleoid with DNA. This is surrounded by the cytoplasm where energy is generated and proteins are synthesized. In viruses, the inner core of genetic material is either DNA or RNA, but they have no cytoplasm and hence depend on the host for their energy and proteins (i.e. they are metabolically inert).

• Reproduction. Bacteria reproduce by binary fission (a parent cell divides into two similar cells), but viruses disassemble, produce copies of their nucleic acid and proteins, and then reassemble to produce another generation of viruses. As viruses are metabolically inert, they must replicate within host cells. Bacteria, however, can replicate extracellularly (except rickettsiae and chlamydiae, which are bacteria that also require living cells for growth).

Eukaryotes and prokaryotes

As mentioned above, another modification of classifying cellular organisms is to divide them into prokaryotes (i.e. Archaea and Bacteria) and eukaryotes (Greek karyon: nucleus). Fungi, protozoa and humans, for instance, are eukaryotic, whereas bacteria are prokaryotic. In prokaryotes, the bacterial genome, or chromosome, is a single, circular molecule of double-stranded DNA, lacking a nuclear membrane (smaller, single or multiple circular DNA molecules called plasmids may also be present in bacteria), whereas the eukaryotic cell has a true nucleus with multiple chromosomes surrounded by a nuclear membrane.

Bacteria comprise the vast majority of human pathogens, while archaea appear rarely to cause human disease and live in extreme environments (e.g. high temperature or salt concentrations). Archaea received little attention traditionally as they cannot be easily cultured in the laboratory. Interestingly, recent studies using novel techniques such as pyrosequencing have uncovered their presence in the oral cavity. Some studies have even shown that certain species of archaea are more frequently found in subgingival plaque in periodontal disease.

Morphology

Shape and size

The shape of a bacterium is determined by its rigid cell wall. Bacteria are classified by shape into three basic groups (Fig. 2.1A and B):

Fig. 2.1 Common bacterial forms. (A) Coccus; (B) capsulated diplococci; (C, D) cocci in chains (e.g. streptococcus) and clusters (e.g. staphylococcus); (E) bacillus; (F, G) capsulated and flagellated bacillus (e.g. Escherichia coli); (H) curved bacilli (e.g. Vibrio spp.); (I) spore-bearing bacilli (e.g. Clostridium tetani); (J) spirochaete.

1. cocci (spherical)

2. bacilli (rod-shaped)

3. spirochaetes (helical).

Some bacteria with variable shapes, appearing both as coccal and bacillary forms, are called pleomorphic (pleo: many; morphic: shaped) in appearance.

The size of bacteria ranges from about 0.2 to 5 µm. The smallest bacteria approximate the size of the largest viruses (poxviruses), whereas the longest bacilli attain the same length as some yeasts and human red blood cells (7 µm).

Arrangement

Bacteria, whichever shape they may be, arrange themselves (usually according to the plane of successive cell division) as pairs (diplococci), chains (streptococci), grape-like clusters (staphylococci) or as angled pairs or palisades (corynebacteria).

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Gram-staining characteristics

In clinical microbiology, bacteria can be classified into two major subgroups according to the staining characteristics of their cell walls. The stain used, called the Gram stain (first developed by a Danish physician, Christian Gram), divides the bacteria into Gram-positive (purple) and Gram-negative (pink) groups. The Gram-staining property of bacteria is useful both for their identification and in the therapy of bacterial infections because, in general, Gram-positive bacteria are more susceptible to penicillins than Gram-negative bacteria.

Structure

The structure of a typical bacterium is shown in Figure 2.2. Bacteria have a rigid cell wall protecting a fluid protoplast comprising a cytoplasmic membrane and a variety of other components (described below).

Fig. 2.2 A bacterial cell.

Structures external to the cell wall

Flagella

Flagella are whip-like filaments that act as propellers and guide the bacteria towards nutritional and other sources (Fig. 2.3). The filaments are composed of many subunits of a single protein, flagellin. Flagella may be located at one end (monotrichous, a single flagellum; lophotrichous, many flagella) or all over the outer surface (peritrichous). Many bacilli (rods) have flagella, but most cocci do not and are therefore non-motile. Spirochaetes move by using a flagellum-like structure called the axial filament, which wraps around the cell to produce an undulating motion.

Fig. 2.3 Photomicrograph of a bacterium showing peritrichous flagella. Note the relative length of the flagella compared with the size of the organism.

Fimbriae and pili

Fimbriae and pili are fine, hair-like filaments, shorter than flagella, that extend from the cell surface. Pili, found mainly on Gram-negative organisms, are composed of subunits of a protein, pilin, and mediate the adhesion of bacteria to receptors on the human cell surface – a necessary first step in the initiation of infection. A specialized type of pilus, the sex pilus, forms the attachment between the male (donor) and the female (recipient) bacteria during conjugation, when genes are transferred from one bacterium to another.

Glycocalyx (slime layer)

The glycocalyx is a polysaccharide coating that covers the outer surfaces of many bacteria and allows the bacteria to adhere firmly to various structures, e.g. oral mucosa, teeth, heart valves and catheters, and contribute to the formation of biofilms. This is especially true in the case of Streptococcus mutans, a major cariogenic organism, which has the ability to produce vast quantities of extracellular polysaccharide in the presence of dietary sugars such as sucrose.

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Capsule

An amorphous, gelatinous layer (usually more substantial than the glycocalyx) surrounds the entire bacterium; it is composed of polysaccharide, and sometimes protein (e.g. anthrax bacillus). The sugar components of the polysaccharide vary in different bacterial species and frequently determine the serological type within a species (e.g. 84 different serological types of Streptococcus pneumoniae can be distinguished by the antigenic differences of the sugars in the polysaccharide capsule). The capsule is important because:

• It mediates the adhesion of bacteria to human tissues or prosthesis such as dentures or implants – a prerequisite for colonization and infection.

• It hinders or inhibits phagocytosis; hence, the presence of a capsule correlates with virulence.

• It helps in laboratory identification of organisms (in the presence of antiserum against the capsular polysaccharide the capsule will swell greatly – a phenomenon called the quellung reaction).

• Its polysaccharides are used as antigens in certain vaccines because they elicit protective antibodies (e.g. polysaccharide vaccine of S. pneumoniae).

Cell wall

The cell wall confers rigidity upon the bacterial cell. It is a multilayered structure outside the cytoplasmic membrane. It is porous and permeable to substances of low molecular weight.

The inner layer of the cell wall is made of peptidoglycan and is covered by an outer membrane that varies in thickness and chemical composition, depending upon the Gram-staining property of the bacteria (Fig. 2.4). The term ‘peptidoglycan’ is derived from the peptides and the sugars (glycan) that make up the molecule. (Synonyms for peptidoglycan are murein and mucopeptide.)

Fig. 2.4 Chemical structure of cross-linking peptidoglycan component of cell wall, common to both Gram-positive and Gram-negative bacteria.

(After Sharon, N (1969). The bacterial cell wall. Scientific American 220, 92.)

The cell walls of Gram-positive and Gram-negative bacteria have important structural and chemical differences (Fig. 2.5):

Fig. 2.5 Structural features of Gram-positive and Gram-negative cell walls.

• The peptidoglycan layer is common to both Gram-positive and Gram-negative bacteria but is much thicker in the Gram-positive bacteria.

• In contrast, the Gram-negative organisms have a complex outer membrane composed of lipopolysaccharide (LPS), lipoprotein and phospholipid. These form porins, through which hydrophilic molecules are transported in and out of the organism. The O antigen of the LPS and the lipid A component are also embedded in the outer membrane. Lying between the outer membrane and the cytoplasmic membrane of Gram-negative bacteria is the periplasmic space. It is in this space that some bacterial species produce enzymes that destroy drugs such as penicillins (e.g. β-lactamases).

• The LPS of Gram-negative bacteria, which is extremely toxic, has been called the endotoxin. (Hence, by definition, endotoxins cannot be produced by Gram-positive bacteria as they do not have LPS in their cell walls.) LPS is bound to the cell surface and is only released when it is lysed. It is responsible for many of the features of disease, such as fever and shock (see Chapter 5).

• The cell walls of some bacteria (e.g. Mycobacterium tuberculosis) contain lipids called mycolic acids, which cannot be Gram-stained, and hence are called acid-fast (i.e. they resist decolourization with acid alcohol after being stained with carbolfuchsin).

Bacteria with defective cell walls

Some bacteria can survive with defective cell walls. These include mycoplasmas, L-forms, spheroplasts and protoplasts.

Mycoplasmas do not possess a cell wall and do not need hypertonic media for their survival. They occur in nature and may cause human disease (e.g. pneumonia).

L-forms are usually produced in the laboratory and may totally or partially lack cell walls. They may be produced in patients treated with penicillin and, like mycoplasmas, can replicate on ordinary media.

Both spheroplasts (derived from Gram-negative bacteria) and protoplasts (derived from Gram-positive bacteria) lack cell walls, cannot replicate on laboratory media and are unstable and osmotically fragile. They require hypertonic conditions for maintenance and are produced in the laboratory by the action of enzymes or antibiotics.

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Cytoplasmic membrane

The cytoplasmic membrane lies just inside the peptidoglycan layer of the cell wall and is a ‘unit membrane’ composed of a phospholipid bilayer similar in appearance to that of eukaryotic cells. However, eukaryotic membranes contain sterols, whereas prokaryotes generally do not (the only exception being mycoplasmas). The membrane has the following major functions:

• active transport and selective diffusion of molecules and solutes in and out of the cell

• electron transport and oxidative phosphorylation, in aerobic species

• synthesis of cell wall precursors

• secretion of enzymes and toxins

• supporting the receptors and other proteins of the chemotactic and sensory transduction systems.

Mesosome

This is a convoluted invagination of the cytoplasmic membrane that functions as the origin of the transverse septum that divides the cell in half during cell division. It is also the binding site of the DNA that will become the genetic material of each daughter cell.

Cytoplasm

The cytoplasm comprises an inner, nucleoid region (composed of DNA), which is surrounded by an amorphous matrix that contains ribosomes, nutrient granules, metabolites and various ions.

Nuclear material or nucleoid

Bacterial DNA comprises a single, supercoiled, circular chromosome that contains about 2000 genes, approximately 1 mm long in the unfolded state. (It is analogous to a single, haploid chromosome.) During cell division, it undergoes semiconservative replication bidirectionally from a fixed point.

Ribosomes

Ribosomes are the sites of protein synthesis. Bacterial ribosomes differ from those of eukaryotic cells in both size and chemical composition. They are organized in units of 70S, compared with eukaryotic ribosomes of 80S. These differences are the basis of the selective action of some antibiotics that inhibit bacterial, but not human, protein β-synthesis.

Cytoplasmic inclusions

The cytoplasm contains different types of inclusions, which serve as sources of stored energy; examples include polymetaphosphate, polysaccharide and β-hydroxybutyrate.

Bacterial spores

Spores are formed in response to adverse conditions by the medically important bacteria that belong to the genus Bacillus (which includes the agent of anthrax) and the genus Clostridium (which includes the agents of tetanus and botulism). These bacteria sporulate (form spores) when nutrients, such as sources of carbon and nitrogen, are scarce (Fig. 2.6). The spore develops at the expense of the vegetative cell and contains bacterial DNA, a small amount of cytoplasm, cell membrane, peptidoglycan, very little water and, most importantly, a thick, keratin-like coat. This coat, which contains a high concentration of calcium dipicolinate, is remarkably resistant to heat, dehydration, radiation and chemicals. Once formed, the spore is metabolically inert and can remain dormant for many years. Spores are called either terminal or subterminal, depending on their position in relation to the cell wall of the bacillus from which they developed.

Fig. 2.6 The cycle of sporulation. (A) Vegetative cell; (B) ingrowth of cytoplasmic membrane; (C) developing forespore; (D) forespore completely cut off from the cell cytoplasm; (E) development of cortex and keratin spore coat; (F) liberation of spore and conversion to vegetative state under favourable conditions.

When appropriate conditions supervene (i.e. water, nutrients), there is enzymatic degradation of the coat, and the spore transforms itself into a metabolizing, reproducing bacterial cell once again (Fig. 2.6).

Clinical relevance of bacterial spores

The clinical importance of spores lies in their extraordinary resistance to heat and chemicals. Because of this, sterilization cannot be easily achieved by boiling; other, more efficacious methods of sterilization, such as steam under pressure (autoclaving), are required to ensure the sterility of products used for surgical purposes (Chapter 37). This property of bacterial spores is exploited when they are used for evaluating the sterilization efficacy of autoclaves; spores of Bacillus stearothermophilus and other species are used for this purpose.

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Taxonomy

The systematic classification and categorization of organisms into ordered groups are called taxonomy. A working knowledge of taxonomy is useful for diagnostic microbiology and for studies in epidemiology and pathogenicity.

As mentioned at the beginning of this chapter, organisms encountered in medical microbiology fall into the domains of Bacteria, Archaea and Eucarya. Although this system of classification is based on the evolutionary relatedness or the genetic homogeneity of the species represented in each domain, a more pragmatic means of classification is employed in the clinical microbiology laboratory. Such bacterial classification is somewhat artificial in that they are categorized according to phenotypic (as opposed to genotypic) features, which facilitate their laboratory identification. These comprise:

• morphology (cocci, bacilli, spirochaetes)

• staining properties (Gram-positive, Gram-negative)

• cultural requirements (aerobic, facultative anaerobic, anaerobic)

• biochemical reactions (saccharolytic and asaccharolytic, according to sugar fermentation reactions)

• antigenic structure (serotypes).

Most of the medically and dentally important bacteria are classified according to their morphology, Gram-staining characteristics and atmospheric requirements. A simple classification of medically important bacteria is given in Figures 2.7 and 2.8.

Fig. 2.7 A simple classification of Gram-positive bacteria.

Fig. 2.8 A simple classification of Gram-negative bacteria.

Genotypic taxonomy

In contrast to the classical phenotypic classification methods outlined above, genotypic classification and speciation of organisms are becoming increasingly important and useful. Genotypic taxonomy exploits the genetic characteristics, which are more stable than the sometimes transient phenotypic features of organisms. These methods essentially evaluate the degree of DNA homology of organisms in order to speciate them, for example by assessing molecular guanine and cytosine (GC) content, ribotyping, random amplification of polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Novel bacterial typing methods based on the nucleotide sequences of ribosomal RNA (rRNA) genes have become a robust way of assessing bacterial identity. Further details of these methods are given in Chapter 3.

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Additionally, recent research indicates that endogenous bacterial habitats in humans, including the oral cavity, harbour a flora that cannot be cultured using routine laboratory techniques. These so-called unculturable species comprise both bacteria and archaea, mentioned above and can only be detected by molecular techniques or metagenomics (e.g. by direct amplification of 16S RNA). The role of these totally new phylotypes of bacteria in either disease or health awaits clarification.

Both the culturable and unculturable organisms in the healthy oral cavity are now given the term ‘core microbiome’. The analysis of this core microbiome has been greatly facilitated by a recently developed technique called pyrosequencing (a method of DNA sequencing). The data from pyrosequencing studies have revealed that the oral cavity in health may contain more than 1000 different bacterial species (see Chapter 31)!

How do organisms get their names?

Organisms are named according to a hierarchical system, beginning with the taxonomic rank domain, followed by kingdom, phylum, class, order, family, genus and species (Table 2.3). The scientific name of an organism is classically a binomial of the last two ranks, i.e. a combination of the generic name followed by the species name, e.g. Streptococcus salivarius (note that the species name does not begin with a capital letter). The name is usually written in italics with the generic name abbreviated (e.g. S. salivarius). When bacterial names are used adjectivally or collectively, the names are not italicized and do not begin with a capital letter (e.g. staphylococcal enzymes, lactobacilli).

Table 2.3 Hierarchical ranks in classification of organisms

Taxonomic rank	Example
Domain	Bacteria
Kingdom	Bacteria
Phylum	Firmicutes
Class	Bacilli
Order	Lactobacillales
Family	Lactobacillaceae
Genus	Lactobacillus
Species	Lactobacillus acidophilus

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Key facts

Note: clinically relevant facts and practice points are italicized; key words are in bold.

• The word ‘microorganism’ (microbe) is used to describe an organism that cannot be seen without the use of a microscope.

• The main groups of microbes are algae, protozoa, fungi, bacteria and viruses, with progressively decreasing size.

• All living cells are either prokaryotic (Archea and Bacteria) or eukaryotic.

• Prokaryotes such as bacteria are simple cells with no internal membranes or organelles.

• Eukaryotes have a nucleus, organelles such as mitochondria and complex internal membranes (e.g. fungi, human cells).

• Bacteria are divided into two major classes according to staining characteristics: Gram-positive (purple) and Gram-negative (pink).

• Structures external to the cell wall of bacteria are flagella (whip-like filaments), fimbriae or pili (fine, short, hair-like filaments), glycocalyx (slime layer) and capsule.

• Flagella are used for movement, the fimbriae and pili for adhesion and the glycocalyx for adhesion, protection and biofilm formation.

• Cell wall peptidoglycan is common to both Gram-positive and Gram-negative bacteria but thicker in the former; it gives rigidity and shape to the organism.

• Peptidoglycan comprises long chains of N-acetylmuramic acid and N-acetylglucosamine cross-linked by peptide side chains and cross-bridges.

• Lipopolysaccharides (LPS) are integral components of the outer membranes of Gram-negative (but not Gram-positive) bacteria; LPS is the endotoxin and therefore Gram-positive bacteria cannot produce endotoxin.

• Cell walls of some bacteria such as the mycobacteria contain lipids (mycolic acids) that are resistant to Gram staining; these bacteria are called acid-fast organisms.

• Bacterial cytoplasm contains chromosomal nuclear material – nucleoid, ribosomes, inclusions/storage granules.

• Spore formation or sporulation is a response to adverse conditions in Bacillus spp. and Clostridium spp.

• Taxonomy (systematic classification of organisms into groups) can be performed according to morphology, staining reactions, cultural requirements, biochemical reactions, antigenic structure and DNA composition.