Microbiology and Virology

Morphology and Structure

Most bacteria are 1 μm in diameter or larger, which means that they are readily visible by light microscopy and conventional bright-field illumination. However, to visualise the internal structures of the cell, the resolving power of an electron microscope is required. Fig. 7.1 is a diagrammatic representation of the internal structures of the prokaryotic cell.

Many bacteria have a capsule or loose slime around the cell wall. This capsule is an important protective mechanism. The ability of organisms such as Staphylococcus epidermidis to produce slime (biofilm) on the surfaces of cannulae results in the protection of the organism from the action of antimicrobial agents, and difficulty in eradicating the organism in catheter-associated sepsis.

The cell wall of bacteria is unique in its composition and plays a structural role. This macromolecule consists of a backbone of N -acetyl-glucosamine and N -acetyl-muramic acid residues linked to polypeptides, polysaccharides and lipids, and is collectively called ‘peptidoglycan’. Peptidoglycan is responsible for the rigidity of the cell wall and maintenance of the characteristic shape of an organism. Gram stain differentiates bacteria into those that take up and retain a complex of crystal violet and iodine, and those that do not. This ability is a function of the cell wall. Gram-positive organisms (stained blue/black) have a cell wall consisting largely of peptidoglycan linked to teichoic acids. In contrast, the cell wall of Gram-negative organisms (usually counterstained pink) is far more complex with an outer membrane of lipoprotein and lipopolysaccharide (LPS; also unique to bacteria), separated from the peptidoglycan layer by the periplasmic space. This arrangement has important consequences for the ability of Gram-negative bacteria to neutralise the activity of certain antimicrobial agents such as the cell wall active β-lactams (e.g. penicillins and cephalosporins) and prevents glycopeptides (vancomycin) from entering the cell and stopping peptidoglycans from being fully synthesised. Peptidoglycan is synthesised with the assistance of transpeptidases, also known as penicillin-binding proteins (PBPs), which are a target for β-lactams. This group of antibacterial agents is therefore acting against a metabolic pathway unique to bacteria, with consequent low toxicity to eukaryotic cells. The presence of β-lactamases in the periplasmic space may result in the bacteria being resistant to these agents. Mycoplasmas are unique among bacteria in not having a rigid cell wall, while the chlamydiae lack peptidoglycan. Not surprisingly, these bacteria are essentially resistant to β-lactams-based antibiotics.

The cell wall of acid-fast bacteria such as the mycobacteria and Nocardia spp. contains a high lipid content. They are difficult to stain by most stains, but a solution of hot phenolic carbol fuchsin, or the fluorochrome auramine, which binds to the lipid, will resist decolouration with sulphuric acid, and stain the organism.

The nucleus is a tightly coiled circular double strand of DNA, which replicates by fission. Other units of straight or circular DNA termed ‘plasmids’ may occur loosely in the cytoplasm. These may code for non-essential features such as antibiotic resistance or ability to ferment certain sugars such as lactose. The ability of bacteria to transfer plasmid DNA between bacteria of the same or different species may result in the spread of antimicrobial resistance (plasmid mediated). Bacteria may also transfer genetic material from the nucleus (the so-called ‘jumping gene’), leading to stable, chromosomally mediated resistance.

Projecting through the cell wall may be flagellae, fimbriae or pili. Flagellae are long whip-like structures associated with motility. Fimbriae form a fringe around bacteria allowing gliding movement. Pili are longer than fimbriae, and more numerous than flagellae. They are associated with conjugation between bacteria of the same or different species, during which the exchange of genetic material, and hence transferable antibiotic resistance, can occur.

Bacteria are morphologically constrained, they are either rod-shaped (bacilli), spherical (cocci), spirillum (actually helical and not a spiral) or vibrios (curved or comma shaped), budding and filamentous (actinomycetes). Cocci may be in chains (e.g. streptococci) or in clusters (e.g. staphylococci). However, in smears, lactobacilli which are morphologically similar may appear to branch, leading to confusion in the evaluation of cervical specimens for actinomycosis. Some members of the Actinobacteria (i.e. the genus Bifidobacteria form ‘Y’-shaped cells). Many members of the Gram-positive bacteria are also able to produce endospores, a highly resistant resting and survival phase, and can be seen in genera such as Bacillus and Clostridium .

Classification and Typing

The classification of bacteria was complicated by the lack of clear-cut morphological relationships between different members, however in the late 1970s Carl Woese and George E. Fox (Woese and Fox, 1977) proposed a new topology for the tree of life based on DNA. For the first time a rationale framework for classifying bacteria had been devised which did not require biochemical tests or morphological phenotypes. It also maintained the Linnean hierarchy of species, genus, family, order, etc. and preserved grouping of organisms with shared characteristics. However, fine characteristics were not discernible, for example, Escherichia coli strains are indistinguishable in phylogenetic trees which are created from the DNA sequences of the small subunit ribosomal RNA (rRNA) gene, also known as the 16 S rRNA gene. This lack of resolution means that it is not possible to determine if you have isolated a pathogen (e.g. E. coli O157) or have a probiotic (e.g. E. coli Nissle (1917)). Such information has obvious clinical implications. Despite this, knowledge of an organism’s classification/taxonomy is, however, important for a number of reasons. It enables communication between scientists, gives a broad picture of how the organism may behave in vitro and in vivo, and may give some indication of the likely efficacy of proposed antimicrobial chemotherapy. With the cost of DNA sequencing plummeting and currently heading towards 0.1 pence or cent per base, whole genome sequencing (WGS) has become the main tool to characterise and classify bacteria. With a WGS project costing around £30 to 40 for the raw sequence (2021), it is possible to obtain a draft genome which shows the main properties and functions of the organism of interest. The use of WGS is also making its way into public health systems for tracking pathogen outbreaks, including viral pathogens and for fast classification of pathogen virulence factors and antibiotic resistance profiles.

The naming of newly discovered bacterial isolates follows the conventional Latin binomial system, which is overseen by an international body that applies strict rules. The genus is always written with a capitalised first letter and followed by the specific epithet commencing with a lower-case letter. Both components are written in italics – thus, Staphylococcus spp. and Staphylococcus aureus . The generic name may be abbreviated after first use, thus S. aureus , or if confusion is likely to arise, Staph. aureus . All other references to the specific bacterial taxonomic lineage (e.g. family names such as ‘Staphylococcaceae’ and order ‘Bacillales’) should also be italicised, however, this practice is not enforced by many journals. Trivial names such as ‘coliform’, or adjectives such as ‘staphylococcal’ or ‘staphylococci’ are not written in italics and are not proper nouns. Table 7.1 is a simple classification of medically important bacteria based on these characteristics.

In addition to a need to classify bacteria, it is often necessary to distinguish between infecting organisms of the same species, for example when trying to trace the source of a staphylococcal outbreak or confirming the chain of infection in a case of alleged sexual abuse. A variety of methods are available, some more applicable to some species than others. With the advent of WGS and DNA-based approaches the majority of strain typing is based on molecular approaches. Where an organism can be isolated to purity, WGS offers a cheap and relatively quick approach for source tracking in nosocomial outbreaks (Quainoo et al., 2017). For organisms which are harder to grow nucleic-acid based approaches are the most feasible assay, and the Center for Disease Control recommends the use of DNA-based approaches to identify and type Chlamydia .

Pathogenesis

The distinction between commensal and pathogenic organisms is far from clear-cut. Indeed, many of the organisms associated with common infections are part of the normal or transient microbiota of the body. Mere isolation of the organism from a specimen does not necessarily equate with disease. Rather isolation of an organism from a site normally considered sterile is more indicative of infection and disease. For example, the presence of E. coli in the small intestine reflects its normal habitat, but its presence in bladder urine indicates a urinary tract infection. Haemophilus influenzae , Streptococcus pneumoniae and Moraxella catarrhalis are all normal inhabitants of the upper respiratory tract, but each are capable of causing lower respiratory tract infection.

All bacterial species complexes have a Dr Jekyll and Mr Hyde persona, with examples of non-pathogenic strains and highly virulent strains. Examples include the plague bacillus, Brucella spp. and Treponema spp., however in general these genera are considered to be predominantly pathogenic. At the other extreme are organisms that are usually referred to as ‘Generally regarded as safe’ or ‘GRAS’ and are considered quite innocuous unless the host’s defences are markedly impaired. These include ‘opportunistic’ organisms, such as Pseudomonas aeruginosa , which are often associated with sepsis in the immunosuppressed. There are also case reports of probiotic species causing pathologies when an opportunity arises (Boumis et al., 2018). Since it is not possible to continuously maintain a biological surface as sterile in an open system, the surface (e.g. an initially sterile burn) will soon become colonised with whatever organisms are in close proximity. In addition, if the biological surface has become selective because of antibiotic administration, the colonising organism is likely to be resistant to that antibiotic. The concept of creating the selective medium is important; it is, after all, what the laboratory does to select a single organism from a mixture – merely an in-vitro version of what the clinician may unwittingly be doing in vivo.

While a breakdown in the host immune system may lead to commensal organisms causing disease, bacteria have evolved a number of mechanisms to enhance their disease-causing potential and allow them to evade the immune system. Resistance to lysis by serum is a feature of the Enterobacteriaceae , associated with the presence of LPS at the cell surface. Initial contact with the host may be facilitated by a variety of adhesions. Once attached, the presence of a capsule, with or without antigenic similarity to the host, or the production of a protective biofilm may protect the organism against the host’s immune system. More sophisticated evasive mechanisms include the production of proteases that cleave IgA, a feature of pathogens invading via mucosal surfaces such as Neisseria spp., or coating with host proteins, such as fibronectin as found in T. pallidum . Chlamydia trachomatis is able to prevent the fusion of lysosomes to the intracellular phagosome containing the infectious elementary body (EB); thus the host protects the invading organism from destruction. To initiate an infection of a clean wound with Staph. aureus , some 10 ⁵ organisms are required. However, the presence of a foreign body, be it traumatic or a medically inserted cannula, reduces the required inoculum by 99% to 10 ³ . Such numbers are small by microbiological standards.

Iron is an important growth factor for many bacteria, which enables them to fix iron-binding proteins either through specific receptors for lactoferrin or transferrin (e.g. Staph. aureus ), or by producing extracellular chelators such as siderophore (e.g. enterobactin from E. coli ). Other extracellular products such as hyaluronidase and the ureases of Proteus spp. and Helicobacter pylori may also contribute to pathogenesis.

Toxin production is important for the ability of many pathogens to cause disease (virulence). These toxins may be found extracellularly as exotoxins or released upon cell death as endotoxins. Exotoxins are a feature of Gram-positive and Gram-negative organisms. Examples of the action of exotoxins include the neuromuscular effects of Clostridium botulinum and Cl. tetani toxins, gastrointestinal symptoms of cholera, E. coli , Shigella spp. and Staph. aureus , and skin necrosis from Staph. aureus . Some toxins require the infection of the bacteria with a phage for expression, such as diphtheria toxin, which affects the heart and lungs, and the erythrogenic toxin of Str. pyogenes (Group A streptococcus). Staphylococcal toxic shock syndrome toxin is a potent pyrogen. Some exotoxins can be formalin fixed to produce toxoids, which are used as vaccines (e.g. tetanus toxoid).

Endotoxin, otherwise known as LPS, is a feature of the Gram-negative cell wall. An important component of LPS is lipid A, which links it to the outer membrane. Lipid A seems to be responsible for the inflammatory responses associated with the endotoxic shock found in severe Gram-negative septicaemia. While the LPS of the Enterobacteriaceae are among some of the most potent triggers of inflammatory responses there are many other endotoxins that are often overlooked including flagellin, peptidoglycan and lipoteichoic acid (Gram-positive cell wall component). Some of these antigens are also triggers of the innate immune system and need to also be considered as they can influence a host’s response to some of the more canonical antigens such as LPS from E. coli .

Specimen Collection

The quality of the specimen is particularly important in microbiology. A poor specimen transported to a laboratory under less-than-ideal conditions could lead to a result that is at best, unhelpful, and, at worst, highly misleading. In general, specimens from sites thought to be infected will be collected for microscopy, culture and antigen or genome sequencing. In addition, serum samples may be sent for antibody determination. While the pressures on a clinician are appreciated, it is important that full clinical details including any current or intended antimicrobial therapy are also provided. Such information informs how clinical microbiology laboratories will undertake tests and interpret results.

Specimens should almost always be taken before treatment is commenced. Sensitive bacteria will not survive in the presence of antibiotics (unless the agent is bacteriostatic rather than bactericidal), and even if clinically resistant may not be recoverable on artificial media. The correct transport medium should always be used for swabs, to maintain the balance of organisms as similar to that observed at the site and time of sampling, and to ensure survival of pathogens. Because organisms will continue to divide at ambient temperature, specimens should be kept at +4°C and transported to the laboratory as soon as possible. Some organisms are highly sensitive to storage conditions. For example, while conventional deep freeze at −20°C is satisfactory for preserving many species, it is lethal to chlamydiae and many viruses, which survive better when stored below −70°C. Some fastidious organisms such as the gonococcus, which do not survive well out of their in vivo niche, should be either direct plated at the bedside or rapidly transported to the laboratory. To increase the likelihood of a positive result, liquid pus should always be preferred to a swab dipped in the pus. Different antigen or genome tests require different collection media, even where the same organism is being detected. It is therefore necessary to check with the laboratory before sending these specimens. If the possibility of sexual abuse arises, it is vital to set up a formal chain of evidence with the laboratory, or the evidence may not be admissible in court.

Culture

The majority of bacteria are still identified by culture on solid agar media in public health and clinical microbiology laboratories, however, there is a slow adoption of culture-independent assays which expedite identification. Using culture means that a minimum of 18 hours will elapse before even presumptive results are available. Microscopy will assist in some cases, but where there is a high abundance of normal microbiota, such as in the respiratory tract, identification of potential pathogens will be challenging and will thus facilitate some form of DNA or biomarker approach. It is never possible to speciate organisms by microscopy. Thus, intracellular Gram-negative cocci are not necessarily synonymous with Neisseria gonorrhoeae and should never be reported as such until confirmatory results are available. Culture of organisms is necessary in most circumstances to define a full picture of the organisms colonising or infecting a particular site. Sites that are considered to contain very low levels of microbial biomass, such as blood and cerebrospinal fluid, should present little problem to the laboratory as any organism cultured ought to be significant. However, the possibility of contamination of the specimen during collection, even under optimal conditions, may make interpretation of the results difficult. The problem is much greater with specimens from a site with a normal microbiota, because, as previously stated, many potentially pathogenic organisms may also be part of the normal microbiota. Further, it is not yet routinely possible to predict sensitivity to antibiotics without exposing actively divided organisms to them. However, developments in shotgun metagenomics offer a promising approach to meet the challenge of providing clinicians with timely and germane information, without the need to culture. Extraction of bacterial DNA for sequencing on third-generation sequencing platforms such as the Nanopore MinION and bioinformatic analyses may facilitate identification of the predominate strain present and its predicted antibiotic susceptibilities, within 5 hours of the sample being delivered to the laboratory.

Antigen Detection

While no microbiological test is 100% sensitive, the specificity of culture approaches 100%. The same may not be true of antigen-detection systems, although even here the tendency is to concentrate on good specificity over sensitivity. This aim is because a false-positive diagnosis is more likely to mislead than a false-negative one. In the latter situation clinical impression will override the negative report from the laboratory. Non-culture detection tests provide two useful functions. First, they may be used in situations where rapid diagnosis has important therapeutic and public health consequences (e.g. meningitis). Second, the tests are useful to diagnose pathogens that are difficult or slow to isolate in the laboratory. A good example of this is in the diagnosis of chlamydial infection. Because of the need for cell culture to isolate the organism, the development of non-culture detection tests has served to highlight the prevalence and importance of the organism, and also to make diagnostic facilities more widely available. The disadvantage is that the tests are of variable sensitivity, and in some hands, specificity is less than optimal. Direct immunofluorescence tests are of good sensitivity but are subjective; in contrast enzyme-immunoassay systems are of high specificity, but generally of lower sensitivity. The importance of this discussion is that, in low prevalence populations, a low sensitivity (around 90%) may lead to a positive predictive value of under 50%. That is, one in two positive results may be a false positive.

Nucleic Acid Detection

Molecular technology has revolutionised diagnostic microbiology. Tests based on the amplification of DNA such as the polymerase chain reaction (PCR) and the closely related ligase chain reaction (LCR) are now established in the routine diagnosis of certain pathogens (e.g. Neisseria meningitidis and C. trachomatis ). However, because of their extreme sensitivity, these techniques are subject to contamination problems. Only validated tests should ever be used for routine diagnostic purposes. Biological inhibitors may reduce the sensitivity of these tests in practice. The use of WGS is becoming much more routine as a tool to classify and characterise both viral and bacterial pathogens. In part this adoption has been accelerated by the ever-decreasing cost of DNA sequencing and the ease of access to second-generation sequencing platforms such as the Illumina HiSeq, Nextseq500 and MiSeq. In the future it will be much more commonplace for public health microbiology laboratories to rely on WGS and bioinformatic tools to classify a bacterial sample rather than culture-based approaches.

Antibody Detection

Antibody detection tests have the theoretical advantage that all that is required is a sample of clotted blood. Unfortunately, in practice, it is unusual for a definitive diagnosis to be made on a single sample of serum. The antibody rise takes a minimum of 10 to 14 days, and in some infections (e.g. chlamydial infections) more than 3 weeks may elapse. The safest criterion for the diagnosis of infection using serology is a greater than fourfold rise in specific antibody titre in at least a pair of sera. The exceptions are diseases where antibodies to the organism in question are rare in the normal population, or the organism cannot be cultured. An example of the former is plague, and of the latter syphilis. In the case of syphilis, several different tests are carried out on a single specimen in an attempt to confirm the treponemal infection, and also to define the stage of the disease.

Normal Microbiota

The relationship between humans and their microbes is complex, and it represents a shared co-evolutionary history. Products synthesised by one organism may assist the growth of another organism, which may in turn produce factors which will protect the host from invasion by extraneous organisms. Constant stimulation of the host immune system by resident bacteria will lead to early recognition and elimination of related, but potentially pathogenic, organisms, as well as contributing to the control of potentially neoplastic host cells by virtue of antigens similar to aberrant host ones. Intestinal microorganisms are capable of synthesising vitamins. The interactions of the various species of organism found on the skin are important for maintaining a healthy integument by production of fatty acids and other substances that inhibit the growth of potential pathogens. Disruption of this delicate balance will result in symptoms; for example, antibiotics that affect the normal gut microbiota will result in a change in the proportion of different bacterial species, with overgrowth of some at the expense of others. This imbalance is manifest by diarrhoea. A more sinister consequence may be the proliferation of Clostridioides difficile , an anaerobic spore-former present in 2% to 3% of the population, leading to toxin-mediated pseudomembranous colitis. However, it was recently discovered that the opportunity for C. difficile to propagate and to cause infection is most probably due to the use of antibiotics that remove the ability of other bacteria in the community to create colonisation resistance. If the functions of these bacteria are re-introduced, for example, through the use of faecal microbiota transplantation, C. difficile can no longer thrive.

The interaction of aerobic organisms with anaerobic organisms is particularly intriguing. The aerobes serve to consume oxygen, thus lowering the oxygen tension (eH) to very low levels and allowing the proliferation of strictly anaerobic organisms. The anaerobes outnumber the aerobes by 10:1 to 100:1 on the skin, rising to over 1000-fold excess in the large intestine. One gram of faeces contains some 10 ⁸ aerobic organisms and 10 ¹¹ anaerobic organisms. Maintenance of the anaerobic gut microbiota is essential for health, and the use of anaerobe-sparing antibiotics (e.g. ciprofloxacin) where indicated is less likely to lead to diarrhoea as a side-effect.

The predominantly Gram-positive resident microbiota of the skin is supplemented by transient organisms, usually from the environment, and often Gram-negative. They are unable to establish themselves but may survive for several hours. This period is long enough for transfer to occur to susceptible individuals via the examining fingers.

Normal Genital Tract Microbiota of Women

Lactobacillus species have long been recognised as being predominant members of the genital tract microbiota. More recently, metataxonomic approaches have indicated that there exists 5 major vaginal bacterial profile types or community state types (CST), 4 of which were dominated by species from the genus Lactobacillus and a fourth CST which is dominated by strictly anaerobic and low levels of lactic acid bacteria. Specifically, CST I, II, II and V are dominated by L. crispatus , L. gasseri , L. iners and L. jensenii respectively, and are considered to be low diversity communities. CST IV is a relatively high diversity community colonised by species from the Prevotella , Megasphaera , Atopobium and Sneathia genera, for example. Several groups have now independently replicated these findings in different patient cohorts and identified interesting correlations between these CSTs and pre-term birth and cervical cancer, thus identifying the potential for interventions that modulate the vaginal microbiota and abrogate these issues.

The normal microbiota of the vagina changes under the influence of circulating oestrogens. The presence of oestrogen leads to an environment rich in glycogen, and the release of glucose via vaginal α-amylase activity favours the growth of lactobacilli and other acid-tolerant organisms. The metabolism of glycogen breakdown products to lactic acid, results in a pH less than 4.5. Other bacteria commonly present include anaerobic cocci, diphtheroids, coagulase-negative staphylococci and α-haemolytic streptococci. In addition, a number of organisms that are also potential pathogens may colonise. These include β-haemolytic streptococci including Str. agalactiae and Actinomyces spp. The balance between health and disease in the vagina is delicate. Factors leading to alteration of this balance will lead to overgrowth of organisms at the expense of the lactobacilli leading to bacterial vaginosis. Specific disease can be caused by yeast-like fungi (e.g. Candida spp.) or infection with the protozoon Trichomonas vaginalis . Gonococcal and chlamydial infections affect the cervix, causing genital discharge. Bacterial vaginosis, gonococcal and chlamydial infections all predispose to ascending infection resulting in endometritis and salpingitis, with the attendant sequelae of ectopic pregnancy or infertility. Bacterial vaginosis also appears to be a factor in the pathogenesis of pre-term labour.

Gram-Positive and Gram-Negative Bacteria

Table 7.2 lists some of the more medically important bacteria. Staph. aureus is distinguished from other staphylococci by production of coagulase. Increasingly, these organisms are proving to be resistant to the anti-staphylococcal β-lactam antibiotics (penicillins and cephalosporins). Such strains are designated methicillin-resistant Staph. aureus (MRSA) after the now obsolete antibiotic used as a laboratory test to detect them. Strains are frequently also multi-resistant, and some are able to spread easily through clinical areas (epidemic MRSA – EMRSA). MRSA are usually no more virulent than other coagulase-positive staphylococci, and frequently colonise wounds and carrier sites. However, when they do cause infection the antibiotic choice is considerably limited compared with methicillin-sensitive strains.

Streptococci are divided into three broad groups based on their haemolysis of horse blood agar. Strains producing partial haemolysis (resulting in a greenish pigmentation of the agar) are termed α-haemolytic. This group comprises a number of commensal strains found particularly on the skin and in the mouth (‘viridans’ streptococci), but they are also important pathogens in deep-seated abscesses and endocarditis. The pneumococci and enterococci ( Enterococcus (Streptococcus) faecalis and Ent. faecium ) are also important members of this group. Pneumococci are showing increasing resistance to penicillin. The enterococci are frequent super-infecting organisms, particularly associated with cephalosporin therapy. Glycopeptides (vancomycin and teicoplanin) are often required to treat enterococcal infection; consequently the emergence of vancomycin- and teicoplanin-resistant strains (VRE) is a major worry. Complete haemolysis is termed β-haemolysis. Organisms in this group are further subdivided into the Lancefield Groups A to O. Some α-haemolytic strains also have Lancefield antigens (e.g. the enterococcus is Lancefield Group D). The major human pathogens are in Groups A, B, C and G. However, members of these four groups may also occur as normal human microbiota. The Group A streptococcus is the most important pathogen ( Str. pyogenes ) and remains fully sensitive to penicillin. The third broad group is the non-haemolytic streptococci, which are commensal organisms, although anaerobic streptococci may cause wound infections.

The corynebacteria are Gram-positive rods widely distributed over the skin and upper respiratory tract. It is important to differentiate rapidly the pathogenic C. diphtheriae strains from the commensals, and to determine whether the former are toxin-producing strains. C. jeikeium strains have achieved some notoriety by their ability to colonise intravenous cannulae, particularly in the immunosuppressed. Strains are frequently multiply resistant, and may require glycopeptide therapy, or removal of the cannula.

Listeria monocytogenes is of particular importance in obstetrics. It is a motile Gram-positive rod widely distributed in nature. The organism is capable of active division at low temperatures (e.g. in display and domestic refrigerators). Depending on regional, occupational and animal exposure, between 5% and 70% of the population carry the organism in the bowel, and strains can be isolated from soil, vegetables, salads and dairy products, and uncooked or partly cooked chicken. Of the 13 serovars, only two are of importance in human disease. Infection in adults is an important cause of meningitis. Maternal infection usually occurs late in pregnancy, and symptoms range from mild ‘flu-like’ to chills, fever and back pain and bacteraemia. Neonates infected during pregnancy are ill at or soon after birth. Symptoms are non-specific, but respiratory distress is common, with bradycardia, jaundice and hepatosplenomegaly; neurological symptoms and skin rashes are also found. The characteristic lesions found in the placenta and at postmortem examination of infected neonates are miliary granulomata with focal necrosis. Routine macroscopic inspection of the placenta to exclude these macroscopic lesions should be encouraged. Intrapartum neonatal infection will lead to predominantly meningitic symptoms with an incubation period of 5 to 7 days.

The only bacteria to show branching are the actinomycetes. These organisms colonise the mouth, gut and female genital tract and may also colonise intrauterine devices. Pelvic actinomycosis is a rare chronic granulomatous disease. The diagnosis can be made by observing the yellow mycelial masses (sulphur granules) in tissue. Symptoms may mimic pelvic neoplasia, and the distinction is important because actinomycosis may be treated with extended courses of appropriate antibiotics such as amoxicillin or co-trimoxazole. Cytologists frequently report Actinomyces -like organisms seen on cervical smears. This statement is not synonymous with actinomycosis. The organisms seen are usually commensal lactobacilli, which are also long Gram-positive rods and may appear to show branching in smears.

Clostridium perfringens is a component of normal bowel microbiota. Resistant spores are produced under certain conditions, which may survive inadequate disinfection or sterilisation. The organism will proliferate in necrotic or poorly perfused tissue, giving rise to gas gangrene. The source is almost always the patient’s own microbiota. C. difficile is also found in the adult normal bowel in small numbers, and in neonates in large numbers (Jangi and Lamont, 2010). Antibiotics lead to an overgrowth of this organism, and production of exotoxins which gives rise to pseudomembranous colitis and toxic megacolon. Practically all antimicrobials may lead to this condition, but it is particularly associated with clindamycin, cephalosporins and more recently ciprofloxacin. Neonatal tetanus may be encountered in areas of poor hygiene, acquired via the umbilical stump wound. Cl. botulinum produces a powerful neurotoxin. The disease in adults results from ingestion of the pre-formed toxin, but neonatal botulism may develop from bacteria growing in the gut.

The Gram-negative cocci of medical importance are contained within the genus Neisseria . Both N. gonorrhoeae and N. meningitidis are fastidious organisms, and care is necessary with specimen collection to ensure that the organisms remain viable. The organisms are usually found within inflammatory exudate cells. N. meningitidis is a common nasopharyngeal commensal, and the commonest bacterial cause of meningitis. Both organisms are capable of causing genital infection. N. gonorrhoeae infects columnar cells; it is therefore a parasite of the cervix, not the vagina. M. catarrhalis strains are usually resistant to penicillins, which may compromise treatment of exacerbations of chronic bronchitis.

The enteric Gram-negative rods comprise a large group of morphologically identical organisms located in the gut. The simplest classification divides them into those that ferment lactose, and those that do not. The lactose fermenters include E. coli, Enterobacter spp. and Klebsiella pneumoniae . The non-lactose fermenters include the enteric pathogens such as Shigella spp. and the salmonellae. There are over 2000 types of salmonella, including enteric fever-causing typhoid and paratyphoid, and the common species associated with food poisoning such as Salmonella typhimurium and Salmonella enteritidis . Other important Gram-negative aerobic bacilli include Pseudomonas spp. and Acinetobacter spp. These are predominantly environmental organisms that will colonise and infect wounds opportunistically – that is, wounds in patients who are debilitated, immunosuppressed or on long-term inappropriate broad-spectrum antibiotics.

The anaerobic Gram-negative bacilli are non-sporing. Although their growth requirements are very precise, they are widely distributed in the body, colonising small-intestine and to a lesser extent the large intestine, oropharynx and vagina. They may contribute to the formation of abscesses in association either with other anaerobes, or with aerobic organisms.

The precise cause of bacterial vaginosis is unknown. However, the effect is a change in the balance of the bacterial species making up the normal microbiota. The normally predominant Gram-positive lactobacilli are replaced by Gram-variable coccobacilli. These organisms characteristically adhere to the squamous cells and are called ‘clue cells’ when seen in vaginal smears. The organisms include the anaerobic Mobiluncus spp. and the microaerophilic Gardnerella vaginalis . The term ‘vaginosis’ implies that there is no inflammation of the vaginal wall, but a fishy smelling, watery vaginal discharge is produced with a pH greater than 5.0.

Spirochaetes, Mycoplasmas, Chlamydiae and Other Bacteria

Treponema pallidum , the spirochaete that causes syphilis, cannot be easily cultivated in the laboratory and needs to be grown with tissue culture cells. It is also serologically indistinguishable from the spirochaetes that cause yaws and pinta. In consequence, the laboratory can only provide evidence of current or past treponemal infection. It cannot diagnose syphilis. This unsatisfactory state means that if there is any doubt as to the cause of serum treponemal antibodies, the patient must be assumed to have active syphilis and be treated accordingly. Syphilis in pregnancy will affect the fetus, resulting in a number of characteristic clinical features such as rashes, snuffles, teeth abnormalities, hepatosplenomegaly, proceeding over months and years to osteochondritis and gummata. Specific treatment at any time in pregnancy will result in a healthy neonate.

Mycoplasmas are widely distributed throughout plants and animals. There are more than a dozen species colonising humans, in the oropharynx, bowel and genital tract. The majority of these strains are commensal, and their role in disease is controversial. Mycoplasma pneumoniae is an important cause of atypical pneumonia. Mycoplasma hominis is found in some 20% of sexually active women and may be associated with bacterial vaginosis and pelvic inflammatory disease (PID); it causes some cases of pyelonephritis. Ureaplasma urealyticum is present in up to 80% of sexually active women. Its role in disease is less clear. Both U. urealyticum and M. hominis have been isolated from chorioamnionitis. Mycoplasma should be considered as a cause of postpartum pyrexia and treatment with tetracyclines considered if the fever does not settle. M. hominis differs from other mycoplasmas infecting humans by being resistant to macrolides (e.g. erythromycin), but sensitive to clindamycin. Mycoplasma genitalium is difficult to isolate in the laboratory for routine purposes, but there is evidence from molecular studies that it plays a role in pelvic inflammatory disease.

The chlamydiae have a complex life cycle, as they are obligate intracellular parasites with a unique life cycle involving an extracellular transport phase – the EB – and an intracellular phase – the reticulate body (RB). The life cycle is about 48 hours, during which the EB is taken up into a phagosome within the host cell, and transforms into a RB. Division of the RB leads to an inclusion full of daughter RBs, which condense to form the much smaller EBs. Release of the EBs by rupture of the host cell allows infection of further cells. The organisms cannot be cultured on artificial media, requiring living cells. This makes their laboratory isolation inconvenient. Culture has for routine purposes been superseded by antigen detection (e.g. direct immunofluorescence or enzyme immunoassay) or by molecular technology using PCR. Serology is of limited use in the diagnosis of acute chlamydial genital infection owing to cross-reaction of C. trachomatis with the commoner respiratory species C. pneumoniae . As with N. gonorrhoeae , C. trachomatis also infects columnar epithelium, and so is found in cervical cells.

Action of Antibiotics

The unique structure of the bacterial cell wall has led to the development of chemotherapeutic agents with specific antibacterial activity and low host toxicity (see also Chapter 12 ). The β-lactam antibiotics comprise two main groups – the penicillins and cephalosporins – each of which contains a large number of members giving an antibacterial spectrum, at least in theory, spanning the bacterial genera of medical importance. Other members of the class include the monobactams and carbapenems (e.g. imipenem). All act selectively on the PBPs unique to the region of the bacterial cell wall. Glycopeptides such as vancomycin and teicoplanin are also important inhibitors of the cell wall construction, preventing incorporation of new units.

The cell membrane structure of all living organisms is very similar, so polymyxins, which are active at the bacterial cell membrane, are toxic to humans and rarely used systemically. The antifungal agents nystatin and amphotericin B act on the unique sterol-containing membrane of fungi but are in themselves also toxic to animals. The azole antifungals block sterol synthesis and are less toxic.

Similarities of the basic metabolic and nucleic acid synthesising pathways of plants, animals, fungi and bacteria also causes problems of selective toxicity. Consequently, it is necessary to exploit differing enzyme affinities or alternative pathways to kill infecting organisms selectively with minimal adverse effects on the host. The 70 S ribosomes of bacteria are different to the 80 S ribosomes of mammals, so that antibiotics affecting bacterial protein synthesis are likely to be ineffective against the host’s mechanism. Examples include the macrolides (e.g. erythromycin) and lincosamides (e.g. clindamycin), tetracyclines, aminoglycosides (e.g. gentamicin), fusidic acid and chloramphenicol.

Antibiotics can also affect nucleic acid synthesis. Differing enzyme affinities ensure that toxicity to humans is minimised. The quinolones inhibit the α-subunit of bacterial DNA gyrase, preventing supercoiling of the DNA. The ansamycins (e.g. rifampicin) inhibit bacterial DNA-dependent RNA polymerase. Bacteria need to synthesise folic acid in the same way as other organisms. Sulphonamides and trimethoprim act at different points along the folic acid pathway. Bacteria must synthesise folic acid, while mammalian cells require pre-formed folate, and hence are not affected by sulphonamides, which inhibit folic acid formation. Further along the pathway, the reduction of dihydrofolate to tetrahydrofolate requires the action of dihydrofolate reductase. Trimethoprim, the anti-protozoal pyrimethamine and the anti-cancer drug methotrexate all act at this site. Selective toxicity reflects selective affinity for the relevant enzyme.

The actual site of action of nitroimidazole drugs such as metronidazole is unknown. However, the active compound is known to be a reduced form of the drug which is produced only at the very low oxygen tension (eH) produced in the cells of anaerobic bacteria. The action of this active form is thought to be against the nucleus.

Bacterial resistance may be mediated by one of four mechanisms:

• 1.

  The antibiotic may not get into cells (e.g. vancomycin and Gram-negative organisms).

• 2.

  It may be rapidly eliminated by efflux mechanisms (e.g. tetracycline resistance).

• 3.

  Enzymes may destroy the antibiotic, such as β-lactamases and aminoglycoside-modifying enzymes.

• 4.

  The target site may be increased, altered or blocked, such as by rifampicin or quinolone resistance.

What is apparent is that the ingenuity of the bacterial cell knows no bounds when it comes to the battle for survival. The antibiotic that has no resistance to it has not yet been discovered. Multi-resistant bacteria are becoming more common, and more difficult or even impossible to treat with currently available drugs.