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Eubacteria (or bacteria) can be subdivided into those that have cell walls and those that do not. The cell wall-free types are known as the mycoplasmas (or mollicutes). Mycoplasmas are a distinct group of prokaryotes containing a small genome and surrounded by a unique cell membrane. The surface structure of a typical mycoplasma cell is shown in Fig. 1.5.

The cytoplasm is surrounded by a lipid bilayer consisting of phospholipids. Phospholipids are molecules made up of fatty acids linked to glycerol and then, via a phosphate group, to an alcohol. Essentially, these molecules form the basic structure of the cell membranes of most bacteria (with the exception of archaea [see section 1.3.4.2]). They contain a hydrophilic (alcohol) end and a hydrophobic (fatty acid) end, which associate to form two layers (known as a bilayer) consisting of an inner hydrophobic core and an outer hydrophilic surface. Mycoplasmas are unique among prokaryotes, as they can also contain other lipids (sterols, such as cholesterol) associated in the lipid core of the membrane; sterols are usually present only in eukaryotes and add rigidity to the cell membrane, which confers greater resistance to extracellular factors than typical bacterial cell membranes do. Proteins may also be present, spanning the membrane or associated with the membrane surface. Glycolipids (polysaccharides linked to surface lipids) have also been reported on cell surfaces, and they are believed to be involved in cell attachment. The reduced genome size is presumably linked to the lack of cell wall metabolism and other biosynthetic pathways (e.g., purine metabolism) that are required in other bacteria. Overall, as mycoplasmas have no cell wall, they are pleomorphic (many shaped) (Table 1.1). They can be commensals or pathogens of plants, humans, and animals (Table 1.7). Mycoplasmas are common contaminants of cell cultures and are also implicated in chronic diseases, such as chronic fatigue syndrome and rheumatoid arthritis. The lack of a protective cell wall may make mycoplasmas more sensitive to drying, heat, and some biocides. Other bacteria (as discussed below) that normally have a cell wall can also be present in a cell wall-free form and are referred to as L or cell wall-deficient forms. These forms have been found in artificial culture media and in infected tissues. They may be stationary forms that can circumvent host defense mechanisms. Examples of described cell wall-deficient forms of bacteria are Helicobacter, Mycobacterium, Pseudomonas, and Brucella. Some are suspected of being involved in autoimmune diseases, such as rheumatoid arthritis (Propionibacterium acnes) and multiple sclerosis (Borrelia mylophora).

TABLE 1.6 Classification of protozoa, based on their motility mechanisms and microscopic morphologies

Classification and organism	Disease(s)	Comments
Flagellates (motility by flagella)
Giardia lamblia	Giardiasis, including dysentery	Trophozoites (~20 μm; shown above) produce cysts, which can survive water chlorination
Trypanosoma gambiense	Sleeping sickness	Transferred in tsetse flies
Leishmania donovani	Leishmaniasis (kala-azar)	Transferred in sand flies
Amebas (motility by flowing cytoplasm, “pseudopodia”)
Entamoeba histolytica	Amebiasis, including dysentery and liver abscesses	The trophozoites reproduce asexually by binary division and can produce cysts, which can be transferred in contaminated food or water (surviving for up to 5 weeks at room temperature)
Acanthamoeba castellanii	Eye infections; associated with contaminated contact lenses	Commonly found free living in water, with two stages in life cycle (trophozoites and cysts)
Ciliates (motility using cilia)
Paramecium spp.	Dysentery	The trophozoite has two types of nuclei and can be up to 60 μm in length
Balantidium coli	Dysentery	Trophozoites can measure up to 150 μm, with transmission via cyst-contaminated meat
Sporozoans, apicomplexans (no specific motility extensions used)
Plasmodium falciparum	Malaria	Complicated life cycle; sporozoites transferred to humans by female mosquitoes
Cryptosporidium parvum	Severe diarrhea	Oocysts have marked resistance to biocides, surviving in water. When ingested, they hatch to release sporozoites. These forms invade cells of the intestine; they can reproduce asexually through two generations and then produce oocysts by sexual reproduction.
Toxoplasma gondii	Toxoplasmosis	The oocysts are formed in the cat intestine and transferred to other animals

FIGURE 1.4 Life cycle of Toxoplasma gondii.

FIGURE 1.5 Simple representation of a mycoplasma cell surface structure.

Bacteria that contain cell walls can be simply classified based on their cell morphology and general reaction to a staining method known as the Gram stain. The Gram stain is used to differentiate between two types of cell wall structures: gram positive and gram negative. Microscopic examination of stained preparations allows further differentiation based on their shape (Table 1.8). However, this is an oversimplification, as bacteria vary widely in their morphologies and staining characteristics; many other methods are used for further differentiation, including assays of oxygen requirements, growth characteristics, and lipid composition; immunoassays; and molecular biological procedures.

TABLE 1.7 Examples of pathogenic mycoplasmas

Type	Example(s)	Significance
Spiroplasma	S. citri	Plant pathogens, insect parasites
Ureaplasma	U. urealyticum	Human parasites, genital tract diseases
Mycoplasma	M. genitalium, M. pneumoniae	Urethritis, atypical pneumonia

The basic structure of cell wall-containing bacteria consists of an outer cell wall and an inner cell membrane surrounding the internal cytoplasm (Fig. 1.6). The cell surface can also contain additional structures, such as pili, flagella, and capsules, depending on the bacterial species and its growth conditions.

The cell membrane is similar to that in mycoplasmas and consists of a phospholipid bilayer (without sterols) and associated proteins. Membrane proteins can be at the interface with the cytoplasm, embedded within the membrane, and/or associated with the external wall of the cell. Examples are some lipoproteins (proteins with lipid groups attached), in which the lipid component allows anchoring to the membrane. The overall structure is fluid but serves as a barrier to contain the cytoplasm and to restrict the passage of nutrients and ions into and out of the cell. Membrane proteins play vital roles in many cellular activities, including transport mechanisms, enzymatic reactions, cell signaling, energy generation, and cell wall synthesis. For this reason, damage to the cell membrane can render bacteria nonviable. The cell wall structures are less similar and can be considered as three basic types: gram-positive, gram-negative, and mycobacterial cell walls (Fig. 1.7). Mycobacteria (not to be confused with the cell wall-free mycoplasmas) are considered separately due to their unique cell wall structure. The cell wall can play an important role in the resistance of bacteria to disinfection (see chapter 8).

TABLE 1.8 General differentiation of types of bacteria based on their microscopic morphologies and reactions to Gram staining

Bacterial structure	Shape	Example(s)
Cocci		Gram positive: Staphylococcus, Streptococcus Gram negative: Neisseria, Veillonella
Bacilli (rods)		Gram positive: Bacillus, Listeria Gram negative: Escherichia, Pseudomonas
Spirals		Gram negative: Treponema, Borrelia
Pleomorphic		Gram negative: Bacteroides Cell-wall-free bacteria, e.g., Mycoplasma

FIGURE 1.6 Basic structure of a bacterial cell, showing the cell surface in greater detail.

A key component of all bacterial cell walls is peptidoglycan, which is a polysaccharide (a polymer of sugar units) of two repeating sugars, N-acetylglucosamine and N-acetylmuramic acid, linked by (β-1,4 glycosidic (sugar-sugar) bonds (Fig. 1.8). The N-acetylmuramic acids have attached tetrapeptides (peptides of four amino acids), which are composed of amino acids such as L-alanine, D-alanine, D-glutamic acid, and lysine (usually in gram-positive bacteria) or diaminopimelic acid (DAP) (usually in gram-negative bacteria). These tetrapeptides cross-link the polysaccharide layers. The exact structure, extent of cross-linking, and thickness of the peptidoglycan vary among bacteria. For example, Escherichia coli (a gram-negative bacterium) tetrapeptides consist of L-alanine, D-glutamic acid, DAP, and D-alanine, and the peptidoglycan is only a minor component of the cell wall (~10%), which is loosely cross-linked. In contrast, the Staphylococcus aureus peptidoglycan has lysine instead of DAP in the tetrapeptide but is also indirectly linked to an adjacent tetrapeptide by a five-amino-acid (glycine) bridge. The peptidoglycan makes up ~90% of the staphylococcal cell wall and is highly cross linked. It is the dense nature of peptidoglycan in the gram-positive cell walls that allows differentiation in the Gram stain. Some archaea have been found to have a similar but distinct peptidoglycan structure present in their cell walls (see section 1.3.4.2).

Overall, the basic structure of a gram-positive bacterium’s cell wall consists of peptidoglycan; however, other proteins and polysaccharides have been described and can be specific to different bacterial species. These include polysaccharides (e.g., the A, B, and C streptococcal polysaccharides), teichoic acids, and teichuronic acids. The teichoic acids are found in the cell walls of many gram-positive bacteria, including those of Bacillus, Staphylococcus, and Lactobacillus. They are polysaccharides based on ribitol or glycerol, with attached sugars and amino acids, and are covalently linked to peptidoglycan. Some may also be bound to the cell membrane and are known as lipoteichoic acids. Other polysaccharides include the teichuronic acids (e.g., in Bacillus), which are also linked to peptidoglycan. Proteins and enzymes are also found attached to the peptidoglycan or otherwise associated with the cell wall; they may be involved in interaction with host tissues, peptidoglycan turnover, cell division, and nutrient acquisition. Finally, the actinomycetes typically stain gram positive, but with a different cell wall structure. Structurally, they resemble fungi, can form hyphae, and produce spores (sporophores) by filament fragmentation; however, the nucleic acid is free in the cytoplasm and the filaments and cell sizes are much smaller than in eukaryotic fungi (see section 1.3.2). Nocardia, as an example, has a tripartite cell wall structure similar to that of mycobacteria (see below), while Streptomyces has a more typical gram-positive cell wall structure consisting of an external peptidoglycan but also contains a major portion of fatty acids. Table 1.9 gives some common examples of gram-positive bacteria.

In general, the cell wall in gram-negative bacteria has a minor peptidoglycan layer directly bound to an external outer membrane by lipoproteins (Fig. 1.7). The area between the inner and outer membranes is known as the periplasm. The periplasm can contain a variety of proteins involved in cellular metabolism or in interactions with the extracellular environment. The outer membrane is essentially similar to the inner, cytoplasmic membrane but, in addition to phospholipids and integral proteins, also contains LPSs. LPS contains a lipid portion (known as lipid A) that forms part of the external surface of the outer membrane, linked to a polysaccharide (containing a core and an O-polymer of sugars); the types of fatty acids and sugars that make up LPS structure vary among gram-negative species. LPSs, in particular the lipid A portions, are also known as endotoxins, which are pyrogenic and play a role in bacterial infections (see section 1.3.7). Similar to the inner membrane, proteins can be found associated through or at the periplasmic or external surface of the outer membrane. An important group of integral proteins are the porins, which form channels to allow the transport of molecules through the outer membrane. Some common examples of gram-negative bacteria are given in Table 1.10.

FIGURE 1.7 Bacterial cell wall structures. The cell membranes are similar structures in all types. Gram-positive bacteria have a large peptidoglycan layer (shown as crossed lines) with associated polysaccharides and proteins. Gram-negative bacteria have a smaller peptidoglycan layer linked to an outer membrane. The mycobacterial cell has a series of covalently linked layers, including the peptidoglycan-, arabinogalactan-, and mycolic acid-containing sections.

FIGURE 1.8 Basic structure of peptidoglycan. Polysaccharides of repeating sugars are cross-linked by peptide bridges. Two different types of peptide bridges, which have been described in gram-positive and gram-negative bacterial cell walls, are shown.

TABLE 1.9 Examples of gram-positive bacteria

General type	Key characteristics	Example(s)	Significance
Gram-positive cocci	Diverse group of gram-positive cocci; nonsporeformers	Enterococcus (e.g., E. faecalis, E. faecium)	Widely distributed in soil, water, and animals; normal flora in lower gastrointestinal tract; often identified as causing urinary tract diseases and wound infections. Vancomycin-resistant strains (VRE) are a concern in hospital-acquired infections.
Lactococcus Staphylococcus (e.g., S. epidermidis, S. aureus)	Found in plant and dairy products; can cause food spoilageCommon human and animal parasites. S. epidermidis is usually found on the skin and mucous membranes. S. aureus is commonly identified as a pathogen, including in skin, wound, gastrointestinal, and lower respiratory tract diseases. Methicillin-resistant S. aureus (MRSA) strains are a leading cause of hospital-acquired wound infections.
Streptococcus (e.g., S. pyogenes, S. pneumoniae)	Common human and animal pathogens. S. pyogenes and S. pneumoniae are both associated with upper and lower respiratory tract diseases, including pharyngitis (sore throat), pneumonia, and scarlet fever. S. pyogenes can also cause a wide variety of other diseases, including skin and soft tissue infections (e.g., cellulitis).
Endosporeforming rods/cocci	Rods or cocci that form dormant, heat-resistant endospores; can be aerobic or anaerobic	Geobacillus	G. stearothermophilus spores are widely regarded as the most resistant to heat and other sterilization methods; used as biological indicators of sterilization efficacy.
Bacillus	Aerobic, rod-shaped bacteria; various strains also used as biological indicators for chemical sterilization processes (e.g., B. atrophaeus, formally known as B. subtilis, for ethylene oxide sterilization); some strains also pathogenic, including B. cereus (food poisoning) and B. anthracis (anthrax in animals/humans);widely distributed and often identified as environmental contaminants
Clostridium	Anaerobic, rod-shaped bacteria; widely distributed, including in soil and water. Some species form part of the normal flora of the human intestine (e.g., C. difficile, which is also a leading cause of hospital-acquired diarrhea). Others can cause wound infections (including C. perfringens and C. tetani, the cause of tetanus) and food poisoning (e.g., C. botulinum).
Regular, non-sporulating rods	Rods, but also other regular forms	Lactobacillus, Listeria	Used in the preparation of fermented dairy products, such as yogurt; widely distributed. L. monocytogenes is a leading cause of food-borne illness, which can cause meningitis and septicemia.
Irregular, non-sporulating rods	Rods, but form irregular shapes	Corynebacterium	Often isolated as human/animal pathogens, in particular, on skin and mucous membranes; C. diphtheriae causes an upper respiratory tract infection with systemic effects (diphtheria) (see Table 1.11). P. acnes is a leading cause of skin acne. Some strains can be found as contaminants in dairy products.
Propionibacterium
Other gram-positive bacteria	Mycobacteria: rods	Mycobacterium	See Table 1.11
Actinomycetes: pleomorphic, including the production of hyphae similar in appearance to those of fungi	Nocardia, Streptomyces	Widely distributed; some are opportunistic pathogens (see Table 1.11)
Some strains produce antibiotics (e.g., streptomycin) and form spores; widely distributed; some pathogenic, including plant pathogens

Some unique gram-negative-staining, obligate intracellular bacteria that were previously thought to be viral in nature have been identified, including the chlamydias and rickettsias. Rickettsias are small bacteria with a simple cell wall structure, similar to gram-negative bacteria, and are pleomorphic in shape (ranging from rods to cocci). Most are transferred to humans by arthropods (ticks and lice). Typical diseases caused by rickettsias include typhus (Rickettsia prowazekii and Rickettsia typhi) and Q fever (Coxiella burnetii). The chlamydias are also small obligate parasites. They are therefore difficult to isolate in vitro, requiring cell culturing, and typically stain as gram-negative coccoid bacteria. They are a serious cause of urogenital infections (Chlamydia trachomatis) and pneumonia (Chlamydophila pneumoniae and Chlamydophila psittaci). Chlamydia cell wall structure is unique; similar to the gram-negative cell wall, it contains an inner and outer membrane and LPS, but it does not appear to have a peptidoglycan layer. As obligate parasites, the cells are very sensitive to heat, drying, and biocides.

Figure 1.6 also shows that other structures can be present on the surfaces of bacteria. Of particular interest in the consideration of biocidal processes are external barriers that can protect the cell from its environment. Many bacteria produce an external layer of highmolecular-weight polysaccharides, as well as associated lipids and proteins, which is referred to as a glycocalyx. This can be a simple, loosely associated slime layer or a more rigid, thicker, and firmly attached capsule structure. Capsules can range in structure and size, typically from one-half to five times the cell diameter in thickness. Glycocalyx production plays an important role in the development of bacterial biofilms, which are a further intrinsic resistance mechanism (see section 8.3.8). Glycocalyx structures are found in both gram-positive and gram-negative bacteria. Examples are Streptococcus mutans (in dental plaque), Streptococcus pneumoniae (in nasopharyngeal colonization), and E. coli (enteropathogenic strains that attach to epithelial cells in the intestine). In addition to direct cell protection, they can also play roles in pathogenesis, in bacterial attachment to surfaces, and in preventing drying of the cell. Other bacteria (including archaea) produce an S-layer, similar to polysaccharide capsules, which is composed of protein and glycoproteins to form an external crystalline structure; an example is the external surface of Bacillus anthracis, which produces an S-layer consisting of two protein types that is itself covered by a unique protein (poly-D-glutamic acid) capsule layer.

TABLE 1.10 Examples of gram-negative bacteria

General type	Key characteristics	Example(s)	Significance
Spirochetes	Thin; helical or spiral shaped	Borrelia	Cause what are often described as tick-borne diseases in animals, humans, and birds (e.g., B. burgdorferi, implicated in Lyme disease)
Treponema	Cause human and animal diseases; T. pallidum causes syphilis, a persistent sexually transmitted disease
Helical, vibroid	Usually mobile; vibroid shaped	Campylobacter Helicobacter	C. jejuni causes gastroenteritis H. pylori causes peptic ulcers due to gastritis
Aerobic or microaerophilic rods and cocci	Diverse group of rods or cocci that use oxygen for growth	Acetobacter Bordetella Legionella	Cause food spoilageB. pertussis causes whooping cough, a respiratory disease Associated with water or moist environments; L. pneumophila causes a form of pneumonia known as Legionnaires’ disease
Neisseria	Most strains are nonpathogenic and found on mucous membranes. N. gonorrhoeae causes the sexually transmitted disease gonorrhoea, and N. meningitidis can cause meningitis in young adults.
Pseudomonas, Burkholderia	Common environmental contaminants in water and soil; some strains are plant pathogens. P. aeruginosa and B. cepacia are frequently implicated in hospital-acquired infections, usually associated with proliferation in moist environments and water lines. Pseudomonads can cause biofilm fouling in industrial water lines.
Facultative anaerobes	Rod-shaped; can grow in the presence or absence of oxygen	Enterobacteriaceae
Erwinia	Plant saprophytes and pathogens
Escherichia	E. coli is the most prevalent microorganism in the lower intestinal tract and a common cause of intestinal and urinary tract infections. It is also widely used as a cloning host in molecular biology.
Salmonella	Leading cause of gastroenteritis, mostly food or water borne; examples are S. enterica serovarTyphi (causing typhoid fever) and S enterica serovarTyphimurium (causing gastroenteritis and enteric fever)
Yersinia	Zoonotic infections; Y. pestis causes plague
Vibrionaceae
Vibrio	Gastrointestinal diseases, including cholera (V. cholerae) and food poisoning (V. parahaemolyticus)
Pasteurellaceae
Haemophilus	Commonly found in the upper respiratory tracts of humans and some animals; H. infiuenzae is a leading cause of meningitis in children
Pasteurella	Can cause septicemia in animals and humans
Other gram-negative bacteria	Various shapes and growth requirements	Bacteroides	Anaerobic rods; commonly found in the intestine and as opportunistic pathogens in wounds
Veillonella	Anaerobic cocci; human and animal parasites
Rickettsia, Chlamydia, Chlamydophila	Obligate intracellular pathogens
Cyanobacteria	Free-living in water; photosynthetic; can be unicellular or filamentous
Myxobacteria	Waterborne bacteria that are motile by a gliding mechanism
Leptothrix	Sheathed, filamentous bacteria associated with polluted water

Bacteria can also have a variety of other proteinaceous cell surface appendages, including pili, fimbriae, and flagella (Fig. 1.6). For example, flagellar filaments are composed primarily of flagellin protein subunits and have other proteins that interact with the cell membrane and/or cell wall structure. Flagella are specifically involved in bacterial motility. Fimbriae and pili play important roles in surface, including cell surface, interactions.

A further cell wall structure that deserves separate consideration is the mycobacterial cell wall (Fig. 1.7). Mycobacteria are aerobic, slow-growing, rod-shaped bacteria (for example, Mycobacterium tuberculosis [Fig. 1.9]), which typically stain gram positive and can be further differentiated by acid-fast stain (staining with fuchsin, which resists acid and alcohol decolorization) due to their unique hydrophobic cell wall structure.

FIGURE 1.9 Cells of Mycobacterium tuberculosis. Courtesy of Clifton Barry, NIAID.

This mycobacterial cell wall structure presents a strong permeability barrier and is responsible for the higher level of resistance to antibiotics and biocides in comparison to other bacteria. The cell membrane is similar to that described in other bacteria, which can be linked to the cell wall by glycolipids. The cell wall has a three-layer structure, consisting of a peptidoglycan layer external to the cell membrane, which is covalently linked to a specific polysaccharide (known as arabinogalactan), and finally an external mycolic acid layer. The peptidoglycan is similar to that in other bacteria, but N-acetylmuramic acid is replaced with N-glycoylmuramic acid and cross-linked by three- and four-amino-acid peptides. Arabinogalactan is a polysaccharide of arabinose and galactose. The mycolic acids are attached to arabinose residues of the arabinogalactan and are some of the longest fatty acids known in nature. In mycobacteria, they typically range in carbon length from C₆₀ to C₉₀ and can make up >50% of the cell weight. In addition, the mycobacterial cell wall can contain a variety of proteins (including enzymes), short-chain fatty acids, waxes, and LPSs. Examples are the LPS lipoarabinomannan, which plays a role in host interactions during M. tuberculosis infections, and porin proteins, with a function in molecule transport similar to that seen in the outer membranes of gram-negative bacteria. In some disease-causing mycobacteria, these may also form an external capsule containing enzymes and adherence factors that play roles in mycobacterial pathogenesis. Similar basic cell wall structures have been identified in other bacteria, including actinomycetes (Nocardia) and gram-positive rods (Corynebacterium), with notably shorter-chained mycolic acids of C₄₆ to C₆₀ and C₂₂ to C₃₂, respectively, and in some cases (Amycolatopsis) no mycolic acids. Examples of bacteria with mycobacterium-like cell wall structures are given in Table 1.11.

TABLE 1.11 Cell wall structures in mycobacteria and related organisms

General type	Key characteristics	Example(s)	Significance
Mycobacteria	Slowly to very slowly growing; acid-fast; generally gram positive; aerobic; rod shaped but also pleomorphic or filamentous	Mycobacterium
• Slowly growing (weeks to months)
M. tuberculosis, M. bovis	Cause tuberculosis, a respiratory tract disease, in humans and animals
M. leprae	Causes leprosy, a skin and nerve disease
M. avium	Ubiquitous in nature, including water, dust, and soil; can cause disease in poultry, swine, and immunocompromised humans
• Rapidly growing (3 to 7 days)
M. chelonae, M. gordonae	Can be found as water contaminants and have been identified as pseudoinfections; some strains show high resistance to some biocides
M. fortuitum	Identified in a variety of immunocompromised patient infections, including wound infections
Actinomycetes	Filamentous; gram negative; pleomorphic	Nocardia	Widely distributed, including in soil; some pathogenic, including N. asteroides in pulmonary and systemic infections in humans
Irregular rods	Irregular rods; gram positive	Corynebacterium	Obligate parasites on skin and mucous membranes; pathogenic strains include C. diphtheriae