Bacteria are unicellular organisms and usually occur in simple shapes such as rods, cocci, spiral forms and occasionally as branching filaments. They typically have rigid cell walls containing a peptidoglycan layer and multiply by binary fission. Bacteria are smaller and less complex than eukaryotic cells and do not contain membrane-bound organelles. Genetic information essential for organism survival, the core genome, is usually contained in a single circular chromosome; a nuclear membrane and a nucleolus are absent. Some bacteria have more than one chromosome and chromosomes in certain bacteria are linear. The accessory genome encodes non-essential cell functions and may include plasmids and bacteriophages (see Chapter 3). Despite their morphological diversity, most bacteria are between 0.5 and 5 μm in length. Motile bacteria possess flagella by which they can move through liquids in vivo and in vitro.

Most bacteria found in nature are not harmful to humans, animals or plants. Some bacteria make an important contribution to the utilization of nutrients in soil, in water and in the digestive tracts of animals. Bacteria which cause disease in animals or humans are referred to as pathogenic bacteria.

A typical bacterial cell is composed of a capsule, cell wall, cell membrane, cytoplasm (containing nuclear material) and appendages such as flagella and pili. Some species of bacteria can produce dormant forms termed spores or endospores, structures which are resistant to environmental influences. The principal structural components of bacterial cells are presented in Table 1.1. Some bacteria can synthesize extracellular polymeric material, termed a capsule, which forms a well-defined structure, closely adherent to the cell wall. In the body, capsules of pathogenic bacteria interfere with phagocytosis. The tough, rigid cell walls of bacteria protect them from mechanical damage and osmotic lysis. Differences in the structure and chemical composition of the cell walls of bacterial species account for variation in their pathogenicity and influence other characteristics, including staining properties. Mycoplasmas, an important group of bacteria, lack rigid cell walls but have a flexible triple-layered outer membrane.

On the basis of colour when stained by the Gram method, bacteria can be divided into two major groups, Gram-positive and Gram-negative; this colour reaction is determined by the composition of the cell wall. Gram-positive bacteria, which stain blue, have a relatively thick uniform cell wall which is composed mainly of peptidoglycan and teichoic acids. In contrast, Gram-negative bacteria, which stain red, have cell walls with a more complex structure, consisting of an outer membrane and a periplasmic space containing a comparatively small amount of peptidoglycan.

The cell membranes of bacterial cells are flexible structures composed of phospholipids and proteins. Active transport of nutrients into the cell and elimination of waste metabolites are functions of the cell membrane and it is also the site of electron transport for bacterial respiration. The cytoplasm, which is enclosed by the cell membrane, is essentially an aqueous fluid containing the nuclear material, ribosomes, nutrients, enzymes and other molecules involved in synthesis, cell maintenance and metabolism.

In most bacteria, the bacterial genome is composed of a single haploid circular chromosome containing double-stranded DNA. Bacterial genomes differ in size depending on the species. Plasmids, small circular pieces of DNA which are separate from the core genome, are capable of autonomous replication. Plasmid DNA may encode characteristics such as antibiotic resistance and exotoxin production. All protein synthesis takes place on ribosomes, structures composed of ribonucleoproteins.

Motile bacteria possess flagella, attached to the cell wall, which are usually several times longer than the bacterial cell and are composed of a protein called flagellin. Fine, straight, hair-like structures called pili or fimbriae, composed of the protein pilin, are attached to the cell wall of many bacteria. In many pathogenic Gram-negative bacteria, adhesins present at the tips of pili function as attachment structures for mammalian cells.

Dormant, highly resistant structures, termed endospores, are formed by some bacteria to ensure survival during adverse environmental conditions. The only genera of pathogenic bacteria which contain endospore-forming species are Bacillus and Clostridium. The resistance of endospores is attributed to their layered structure, their dehydrated state, their negligible metabolic activity and their high content of dipicolinic acid. Because endospores are thermostable, moist heat at 121°C for 15 minutes is required for their inactivation.

Appropriate conditions relating to moisture, pH, temperature, osmotic pressure, atmosphere and nutrients are required for bacterial growth. Bacteria replicate by binary fission. The generation time, the length of time required for a single bacterial cell to yield two daughter cells, ranges from 30 minutes to 20 hours. Long-term preservation of microorganisms usually involves freezing procedures. Heat treatment or chemicals can be used for inactivation of bacteria.

Following inoculation of bacterial cells into fresh broth medium, the growth curve of the culture exhibits lag, exponential and stationary phases and a final decline phase. During the lag phase, bacterial cells are metabolically active but not dividing; binary fission of cells results in an exponential increase in numbers. Procedures which can be used for total cell counting include direct microscopy, electronic methods and real-time quantitative polymerase chain reaction (PCR)-based methods. Viable bacterial numbers can be determined by colony counting and by membrane filtration. Accurate cell counts may be required for specific purposes such as vaccine preparation and for bacteriological testing of water.

Bacteria acquire nutrients from their immediate environment. Nutrient media for the isolation of pathogenic bacteria are formulated to supply particular growth factors for particular groups of organisms. Most bacteria require carbon and nitrogen in relatively large amounts. Trace elements and certain growth factors such as vitamins are also essential for bacterial growth.

In addition to nutritional factors, growth of bacteria is influenced by genetic factors and by chemical, physical and other environmental influences. Growth of bacteria in culture is influenced by temperature, hydrogen ion concentration, availability of moisture, atmospheric composition and osmotic pressure. Most bacteria grow optimally at neutral pH. The majority of pathogenic bacteria can be grown aerobically on a nutrient agar medium at 37°C, close to the body temperature of humans and most domestic species. Bacteria with an optimal incubation temperature of 37°C are termed mesophiles and most pathogenic bacteria belong to this category. Based on their preference for particular levels of oxygen, bacteria can be assigned to four main groups, namely aerobes, anaerobes, facultative anaerobes and microaerophiles. A fifth group, capnophiles, are aerobic bacteria with a requirement for carbon dioxide. Anaerobic bacteria are unable to grow in an atmosphere containing oxygen. Strict anaerobes are cultured in tightly sealed jars in an atmosphere from which free oxygen has been removed.

Subculturing can be used for the short-term preservation of bacteria. Limitations of this procedure include death of some cells and a risk of contamination and mutation. Long-term methods of preservation include freeze-drying (lyophilization), freezing at –70°C and ultra-freezing in liquid nitrogen at –196°C. Freezing organisms in vials containing 20–30 porous polypropylene beads which can be removed and cultured singly is a convenient method of avoiding the need for repeated freezing and thawing of cultures. If properly used, these preservation methods can maintain organisms in a hypobiotic state for more than 30 years and ensure that the organisms remain unchanged and uncontaminated.

Sterilization is the method employed for the destruction of microorganisms on equipment used in microbiological and surgical procedures. Physical and chemical methods can be used for inactivation of microorganisms. Chemicals which inactivate bacteria include disinfectants and other compounds with bactericidal activity. Methods for preventing spoilage or limiting microbial growth in food are presented in Table 2.1. Physical methods for sterilizing equipment or fluids are presented in Table 2.2. Sterilization procedures are effective for the destruction of bacterial, fungal and viral agents. When dealing with bacterial endospores, such as those of Clostridium species, heating at a temperature of 121°C for 15 minutes in moist heat is required for their inactivation.

Much of the genetic information in bacteria is contained on a single chromosome located in the cytoplasm of the cell. Bacterial genomes differ in size and express characteristic traits or phenotypes.

Properties of a bacterial cell, including those of veterinary interest such as antimicrobial resistance and virulence, are determined by the microbial genome. The genomic structure consists of three types of genetic information, the chromosome, plasmids and bacteriophages. A typical bacterium consists of a core genome, mainly composed of genes located on the chromosome consisting of double-stranded DNA, and an accessory genome comprising plasmid and bacteriophage DNA. In Escherichia coli K-12, the chromosome is a circular double-stranded DNA molecule of approximately 4.6 × 10⁶ base pairs, containing 157 RNA-encoding genes including ribosomal and transfer RNA along with open reading frames (ORFs) coding for 4,126 bacterial proteins. Bacterial chromosomes typically contain sufficient DNA to encode between 1,000 and 4,000 different genes. Individual genes consist of a starting point, referred to as the start codon and composed of the nucleotides ATG, an ORF and a stop codon (TTA, TAG or TGA).

Although the bacterial chromosome exists free in the cytoplasm, it is compacted through supercoiling and looping of its structure. The central tenets of genetics consist of the expression of a gene from its locus on the chromosome or on a plasmid through transcription (production of messenger RNA or mRNA synthesis) and finally translation, decoding of the mRNA to produce a polypeptide. As the DNA is located in the bacterial cytoplasm, this facilitates the simultaneous transcription and translation of bacterial genes. The gene sequence and its subsequent expression through diverse biochemical pathways accounts for the phenotypic variation observed among bacteria. Recently, these specialized topics have given rise to defined areas of research, referred to as genomics, functional genomics or transcriptomics, and proteomics.

Bacteria replicate by binary fission and the daughter cells produced are usually indistinguishable genetically. Replication of the chromosome in bacteria begins at a specific location referred to as the origin of replication (or origin), at a locus referred to as ori. The two parental strands of the helical DNA unwind under the influence of the enzyme DNA helicase and two identical helical DNA molecules are formed through the action of the replicating enzyme, DNA polymerase. The ends of the newly synthesized strands are joined by DNA ligase, resulting in circular chromosomes.

Transcription and translation, the expression of genetic information

Transcription is an enzyme-mediated process that involves DNA being copied from the positive strand, forming an mRNA molecule. This process is mediated by the enzyme DNA-dependent RNA polymerase that binds to the promoter region of a gene, which is composed of two conserved DNA-binding sites referred to as the –35 and –10 promoter sequences. The two strands of DNA are partially unwound, and locally separate, following which mRNA is synthesized. The information encoded in the mRNA is translated into protein on a ribosome through the involvement of transfer RNA (tRNA), which delivers specific amino acids to the mRNA on the ribosome where the amino acids are enzymatically joined together, forming a peptide bond and extending the polypeptide chain.

Genetic variation may occur following mutation in which a change occurs in the nucleotide sequence of a gene, or by recombination, whereby new groups of genes are introduced into the genome. A stable inheritable alteration in any genome is termed a mutation. A bacterium carrying a mutation is referred to as a mutant. When the original parent and mutant are compared, their genotypes differ and their phenotype may also differ depending on the nature of the mutation. Spontaneous mutations are the result of rare mistakes in DNA replication and occur at a frequency of about one in every 10⁶ cell divisions. Because a gene with altered base pairs may code incorrectly for an amino acid in a protein, the mutation introduced may result in a phenotypic change that may be beneficial or harmful for the organism. Under defined environmental conditions, selected mutations may provide a growth advantage for the mutant over the parent or wild-type bacterium. Mutations can also be experimentally induced by physical, chemical or biological mutagens.

Many viruses that infect animals have RNA genomes which may also undergo mutation. The spontaneous mutation rate associated with these genomes is approximately 1,000-fold higher than that occurring in the host chromosome.

DNA may become damaged following contact with mutagenic chemicals, exposure to UV irradiation and by other means. Different mechanisms are available within the cell to organize the repair of damaged DNA and the choice of the appropriate method depends on the type of damage requiring correction.

Recombination occurs when sequences of DNA from two separate sources are integrated. In bacteria, recombination induces an unexpected inheritable change due to the introduction of new genetic material from a different cell. This new genetic material may be introduced by conjugation, transduction or transformation.

The transfer of genetic material in the form of plasmids of various sizes during conjugation is a complex process that has been extensively studied in the enteric bacterium Escherichia coli. During conjugation, F⁺ (male) bacteria synthesize a modified pilus, the F or sex pilus. This pilus allows direct contact to occur between the male (F⁺) and a suitable female (F^–) bacterium during the process and provides a conduit through which a plasmid or an F-factor can be transferred. One strand of plasmid DNA is unwound and passed to the recipient female (F^–) bacterium in which a complementary strand is later synthesized. Once a new plasmid is formed, the recipient cell is converted into an F⁺ bacterium. Individual bacteria may contain several different types of compatible plasmids.

Plasmid transfer by conjugation has important ecological significance, particularly when antibiotic resistance-encoding genes are involved. A plasmid containing an antibiotic resistance gene in a bacterial cell can, under appropriate conditions, convert the amenable bacterial population into similar plasmid-containing bacterial cells.

DNA acquired either from the original bacterial chromosome or plasmid in a previously infected bacterial cell can be incorporated into phage nucleic acid and transferred by progeny of the phage to susceptible recipient cells in a process called transduction.

Transformation is a process involving the transfer of free or ‘naked’ DNA containing genes on a segment of chromosomal or plasmid DNA from a lysed donor bacterium to a competent recipient. Natural transformation is uncommon and occurs only in a few bacterial genera.

Examples of mobile genetic elements

Insertion sequences and transposons

Transposons are genetic elements that can move as a single unit from one replicon (chromosome, plasmid or bacteriophage) to another. This process is referred to as transposition. Transposons do not possess an origin of replication and consequently replicate as the bacterial host replicates. Transposons encode the necessary features to promote self-mobilization. An example of a simple transposon is an insertion sequence element, denoted as IS, that contains only a transposase-encoding gene required for insertion into new locations. Several IS elements are known and these differ in the numbers of nucleotides they contain. Many bacteria possess multiple IS copies inserted at different locations throughout their genomes.

Some transposons consist of a gene encoding resistance to an antibiotic such as kanamycin, flanked by two IS50 elements, IS50L and IS50R, as in Tn5. Other transposons such as Tn3 encode a β-lactamase gene along with other transposase genes (tnpA and tnpR) required to catalyse the molecular events involved in integration. The complex transposon Tn1546 encodes genes conferring resistance to the glycopeptide antibiotics vancomycin, teicoplanin and the formerly used growth promoter avoparcin.

Integrons are derived from transposon Tn21 and these elements can capture antibiotic resistance, encoding genes via an integron-encoded integrase (a member of the bacterial integrase superfamily) that catalyses a site-specific recombination. These integrons possess a conserved structure (CS) on the proximal end (known as the 5′-CS) containing an integrase gene (int1), a recombination site (att1) and a promoter (P_ant), along with a conserved distal region (3′-CS) containing a qacEΔ1 [conferring resistance to quaternary ammonium compound(s), which are used as disinfectants] and a sul1 determinant conferring resistance to sulphonamides. These CS regions flank a variable central locus into which gene cassettes are recombined. Gene cassettes are composed of one or more ORFs encoding antibiotic resistance gene(s) and a 59-base recognition sequence located at their 3′-end.

Integrons capture a variety of genes encoding resistance to antibiotics such as aminoglycosides and β-lactams, among others, and contribute to the mobilization of these integrons in response to environmental selective pressure. Some integrons possess multiple gene cassettes arranged in a classical ‘head-to-tail’ orientation.

Genetic engineering of bacteria in the laboratory

Useful genetic characteristics encoded by genes in the DNA of a naturally occurring organism can be cloned into a host bacterium in the laboratory, in a process referred to as genetic engineering. These genes can be inserted into cloning vectors, forming recombinant plasmids. They can then be introduced into bacterial cells (usually by transformation) and propagated. The DNA fragments carrying the genes that are selected are produced by either cleaving the donor DNA containing them, using suitable restriction endonuclease enzymes, or through direct amplification by the polymerase chain reaction (see Chapter 4).

Genetic engineering is currently used for the production of vaccines, hormones and other pharmaceutical products (see Chapter 78). Vaccines produced in this manner are potentially safer than conventional vaccines. The genes that code for the vaccine antigens can be cloned separately from genes associated with the parent organism. Genetically engineered vaccines may therefore stimulate an effective immune response without the risk of introducing a pathogen capable of replicating in animals which are being vaccinated.

Genetic databases and bioinformatics

In 1977, the entire DNA sequence of the phage ΦX174 was first published. Since that time there has been an exponential increase in DNA sequence information submitted to gene databases around the world. With increasing volumes of data entries, including high-throughput whole genome sequences for bacteria and other microorganisms, the first of which was Haemophilus influenzae (1.8 Mbp) completed in 1995, it has become impractical to analyse by manual methods these vast amounts of data. This has necessitated the development of specific computational tools to analyse DNA information and identify genes and their corresponding protein sequences, along with regulatory features, at a molecular level.

Bioinformatics is a new scientific discipline that relates to the development of computer algorithms and statistical techniques for analysing and managing genetic information. These tools facilitate the rapid annotation of genome sequences with identification of the position of ORFs within the genome, leading to the identification of genes encoding virulence factors associated with disease production.

Companies involved in the manufacture of pharmaceutical and diagnostic reagents often use bioinformatics to ‘data mine’ genomes, in an attempt to identify new therapeutic agents or useful diagnostic markers.

Most of the characteristics of living bacteria are associated with the genes present on their bacterial chromosome. This structure consists of a double-stranded DNA helix, with all the properties required to control replication of the bacterium, store its genetic information and express some characteristics unique to the organism. All these properties are controlled by specific enzymes and the genetic message is, in some instances, subsequently decoded in a process involving other enzymes, leading to the synthesis of a bacterial protein (see Chapter 3).

The properties of DNA used for analytical purposes derive from its chemical structure. These have been used to develop many of the modern protocols for detection of bacterial pathogens in clinical specimens. A DNA molecule has three important analytical features that facilitate its utilization as a diagnostic target:

• Recognition properties: base-pairing rules in DNA underpin the analytical approach of molecular techniques including DNA probe hybridization, DNA sequencing, the polymerase chain reaction (PCR) and, more recently, microarrays.
• Stability and robust flexibility: the DNA molecule is inherently stable, which facilitates its recovery from degraded material.
• Sequence features: when the DNA sequence of any cell is closely examined, open reading frames (ORFs) encoding genes and other features can be determined.

Molecular hybridization

Any labelled DNA probe, under suitable experimental conditions, would be capable of binding or hybridizing to its complementary strand in solution (based on the DNA base-pairing rules). It is this binding event that is subsequently detected. Examples of molecular hybridization techniques include Southern and northern blotting.

DNA sequencing is the most powerful analytical/diagnostic approach that exists in the molecular armoury. Insight into the understanding of any DNA molecule derives from its nucleotide sequence. The nucleotide sequence can be used to deduce the primary protein structure of the corresponding protein which can subsequently be compared with similar sequences from other organisms. DNA-binding sites and other regulatory features of genes can also be identified.

The DNA sequence of a gene, or the ORF, can be determined using either a chemical- or an enzyme-based approach. The technical principles of the latter method on which modern dideoxy DNA sequencing protocols are based involves the partial replication of a short DNA sequence using all four deoxyribonucleotides (dNTP) and a chemically modified dideoxyribonucleotide (ddNTP) lacking a hydroxyl group at the 2′-carbon on the ribose sugar ring. Like hybridization, this method is based on sequence recognition according to the base-pairing rules and accurate enzymatic synthesis, all of which are features of the naturally occurring replication event. To sequence a DNA molecule the following steps are usually required: primer hybridization, sequence reaction, detection and data analysis.

In a later development of this technology, fluorescent-based automated DNA sequencing was designed to reduce the manual manipulations involved whilst increasing sample throughput. More recent advances in DNA sequencing technology have produced instrumentation capable of sequencing a bacterial genome in a few hours (see Chapter 6).

The PCR assay was developed out of the strategies used for DNA sequencing. Typically a PCR protocol consists of three repeated steps, resulting in the amplification of a discrete segment of DNA (or RNA, after the addition of a reverse-transcription step, see following paragraphs). In the first of these, the template DNA, which has been recovered from a crude preparation of genomic DNA isolated from a microbial pathogen of veterinary interest or from blood or other tissue samples, is denatured, separating the two DNA strands. This is followed by an annealing step, wherein the reaction temperature is lowered, allowing two synthetic DNA primers or oligonucleotides to bind (hybridize) to the template. These primers are located on opposite DNA strands. Finally, the temperature is increased again (typically, 74°C) and a thermostable DNA polymerase enzyme begins a round of synthesis. These steps constitute one cycle and in a conventional PCR reaction up to 30 such cycles are carried out. This repetitive cycling between temperatures facilitates the amplification of a specific DNA target by up to one million-fold.

A programmable thermal cycler controls the rate of temperature change, length of incubation at each temperature and the number of times each cycle is repeated. Multiple cycles produce an amplified PCR product, or amplicon, that can be detected by conventional agarose gel electrophoresis, stained with ethidium bromide and visualized using ultraviolet light.

Conventional PCR-based assays have been developed to detect a broad range of target genes in pathogenic bacteria associated with animals, including food-borne zoonotic pathogens. Commercial kits are also available for these and other pathogenic organisms.

A potential limitation of DNA-based diagnostic methods is that they detect both viable and non-viable bacterial cells. This limitation can be overcome either by using an enrichment step before nucleic acid extraction or by performing an RNA-based detection method using reverse transcriptase (RT)-mediated PCR, in a protocol known as RT-PCR. These assays can also be used to detect RNA deriving from viruses such as rotavirus, coronavirus and norovirus.

Detection and simultaneous quantification of amplicons in real time is an important enabling technology in molecular diagnostics. The method facilitates the determination of the absolute number of a specific DNA target, such as a virulence gene of veterinary importance, relative to a housekeeping gene, such as 16S rRNA, within a living cell. Quantitative real-time PCR (qPCR) can be used to quantify bacteria, other microorganisms and individual genes. Real-time PCR uses fluorescence to detect the presence or absence of a particular DNA or RNA target. It is this detection process that differentiates real-time from conventional PCR.

Expression of any gene in a microorganism or other cell can be determined by measuring the mRNA transcription, using RT-PCR. This technique is referred to as quantitative RT-PCR (qRT-PCR). Based on this protocol, commercial kits are now available to detect and quantify a range of pathogenic organisms relevant to veterinary medicine.

The development of DNA microarrays is based on the use of a solid support to which a series of genes or chemically synthesized segments of those genes can be attached. DNA microarrays can be used in several ways. The arrays can provide useful information for identifying those genes controlling growth of an organism under defined culturing conditions, including aerobic versus anaerobic environmental conditions. In environmental microbiology, DNA microarrays containing 16S rDNA sequences can be used to identify bacteria and other microorganisms present in a particular environment. This DNA microarray is termed a phylochip. Comparative genome analysis makes use of DNA microarrays to compare the gene index of different serovars of Salmonella. DNA chips have been developed to aid in the simultaneous identification of a number of important pathogens including bacteria and viruses that may share similar environmental niches.