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Essential Microbiology For Dentistry 5Th Edition Edition Lakshman Samaranayake Full Chapter
Essential Microbiology For Dentistry 5Th Edition Edition Lakshman Samaranayake Full Chapter
Essential Microbiology For Dentistry 5Th Edition Edition Lakshman Samaranayake Full Chapter
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Essential Microbiology for Dentistry
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Essential
Microbiology for
Dentistry
Fifth Edition
Lakshman Samaranayake
DSc(hc) DDS(Glas) FRCPath FDSRCS(Ed) FDS RCPS(Glas) FRACDS FHKCPath FCDSHK
Vice-Dean, College of Dental Medicine, University of Sharjah, UAE
Professor Emeritus & Immediate-Past Dean of Dentistry, University of Hong Kong, Hong Kong
Honorary Professor & Immediate-Past Head, School of Dentistry, University of Queensland,
Australia
King James IV Professor, Royal College of Surgeons of Edinburgh, UK (2013)
ISBN 9780702074356
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Practitioners and researchers must always rely on their own experience and knowledge
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herein. Because of rapid advances in the medical sciences, in particular, independent
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Contents
Preface vii 20. Chlamydiae, rickettsiae and mycoplasmas 171
Online Study for Students viii 21. Viruses of relevance to dentistry 175
1. Why study microbiology? 1 22. Fungi of relevance to dentistry 187
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Preface
Welcome to the fifth edition of Essential Microbiology for Dentistry! Of course, a tome of this nature cannot be produced without
It is now 22 years since the first edition of this tome was the help of many friends and colleagues. The legacy authors
published in 1996, and since then, the science of microbi- of the Immunology Section (Part 2) were Dr Brian Jones and
omics and infectious diseases has advanced in leaps and Professor Liwei Lu, from the University of Hong Kong, while
bounds. The two major reasons for these transformational Professor Glen C Ulett of Griffith University, Australia expanded
changes have been the exploding new technology that and embellished these chapters as well as other sections of the
delivers novel tools for the identification and reclassification tome. To them, my extreme gratitude.
of organisms, and the emergence of new organisms, espe- Once again, I am indebted to the following colleagues
cially the viruses that change the landscape of dental and worldwide, who graciously permitted the reproduction of their
medical practice. For instance, next generation sequencing work: Professor H Jenkinson, University of Bristol, UK (Fig.
(NGS) technology has revolutionized the field of microbial 3.9); Dr Bernard Low, Malaysia (Fig. 5.1); Professor Willie van
taxonomy and identification of, in particular, the uncultiva- Heerden, University of Pretoria, South Africa (Figs 18.4 and
ble organisms, leading to a radical rethink on the quantity 19.1); Dr Maribasappa Karched of Kuwait University (Fig. 31.2);
and quality of the flora that inhabit our body, including Dr Leanor Haley, CDC, Atlanta, USA (Fig. 22.5); Dr Annette
the oral cavity. In this, the fifth edition of this book, I have Motte, Free University of Berlin, Germany (Fig. 31.8); and
attempted to incorporate the new data as much as possible Professor Saso Ivanowski, Griffith University, Australia (Fig.
while maintaining its popular concise, yet comprehensive 33.8). Figures 38.1 and 38.5 are reproduced from UK Health
outlook. Technical Memorandum No. 01-05, 2009, with permission from
The fact that you are now reading the fifth edition of the Crown Copyright.
book is testimony to its popularity, with more than 40,000 As always, the publishing team at Elsevier led by Martin
copies sold in all five continents; Chinese, Polish and Korean Mellor, Alison Taylor and Helen Leng has pushed me to beat
translations as well as Middle East Editions (Al-Farabi Version) the deadlines despite my myriad duties. Their professionalism
of the book are now in print, although the e-print of the book and patience has my admiration and gratitude. Last but not
appears to be outstripping the hard copy sales. For this, I am least, Hemamali, Dilani and Asanka have lost some quality
deeply grateful to the microbiology teachers in dental schools/ family time due to this tome, and I am eternally grateful to
colleges, as well as the undergraduates and the postgraduates them for their tolerance and understanding.
who are avid fans worldwide. Above all, YOU, the reader, are my most important friend
In compiling this completely revised fifth edition, I have and critic! The many features of this edition are due to your
retained the popular features of the last few editions. One feedback over two decades, and I truly hope that the current
major feature of this edition is an expanded section on infection edition is the finest product thus far. Nevertheless, no book
control (Part 6), which I co-edited with Dr Caroline Pankhurst, is perfect—so please keep on sending your comments, either
of University of London, UK. Other novel additional features good or bad, to me at lakshman@hku.hk.
are sections on NGS technology; the oral microbiome and the
microbiota; endodontic infections; implant-related infections; Lakshman Samaranayake
plaque biofilms and the systemic disease axis and the current Hong Kong
guidelines on antimicrobial prophylaxis. August 2017
Online Study for Students
The latest edition of Essential Microbiology for Dentistry comes Designed to perfectly complement the fifth edition of Essential
with over 300 online MCQs that aim to reinforce the student’s Microbiology for Dentistry, readers are encouraged to work
knowledge as well as provide exam practice for both under- through each module at their own pace to achieve an overall
graduate training and the post-graduate exams set by the UK percentage score at the end of each exercise. This way, they
Royal Colleges and other similar international bodies. can see their grades at a glance across the range of topics to
Reflecting and mirroring the structure of the main textbook, show instantly their areas of strength and those that require
each online learning module presents a mini-series of multiple a revision. When ready, readers can reset the program and
choice questions covering a range of topics which vary from repeat the process with the aim of resitting each module to
microbial structure and taxonomy, to physiology and genet- raise their overall score.
ics. Different classes of microbes are sequentially explored as Prepared by Professor Lakshman Samaranayake, the ques-
well as the host immune response, and the role of effective tions are designed to provide revision and exam practice in
chemotherapy. Downstream modules related to the importance a relaxed, non-pressurised environment with the overall aim
of systemic disease, principles of infection control and infection of improving real exam grades.
control procedures finally complete the picture. The number To access this helpful online self-assessment tool, please log
of questions in each module serve to reflect the importance of in at https://evolve.elsevier.com/Samaranayake/essential/
the topic to dentistry, either in terms of common, and direct,
relevance or by the potential seriousness of its impact on any
individual patient.
1
Why study microbiology?
Microbiology (Greek: mīkros small; bios life), so called because the clinical environment, not only to implement infection control
it primarily deals with organisms too small for the naked eye measures but also to advise the dental team (dental surgery
to see, encompasses the study of organisms that cause disease, assistants, dental hygienists and other ancillary personnel) and
the host response to infection and ways in which such infection to allay patients’ unfounded fears. For all these and many
may be prevented. For our purposes the subject can be broadly other reasons, which the student will discover in the text, the
classified into general, medical and oral microbiology. discipline of microbiology is intimately woven into the fabric
Dental students need both a basic understanding of general of dentistry and composes a crucial component of the dental
and medical microbiology, and a detailed knowledge of clinical curriculum.
oral microbiology in order to diagnose oral microbial infections, It should also be realized that new microbial diseases emerge
which are intimately related to the overall treatment plan for incessantly (due to reasons given in the following section), and
their patients. Moreover, the two major oral disorders—caries the book you are now reading is a primer for understanding
and periodontal disease—that the dental practitioner is fre- and managing such future scenarios, especially in the context
quently called upon to treat are due to changes in the oral of infection control.
bacterial ecosystem and the constituent oral microbiome, and a
grasp of these disease processes is essential for their appropriate
management. A note on emerging and
The impact of these infections on the health and welfare of
the community is simply astonishing. For instance, caries has re-emerging infections
gained the dubious distinction of being the most prevalent
Infectious agents have been adversaries of humans for mil-
disease, affecting almost half (44%) of the world population
lennia. Diseases such as plague have wiped out civilizations
in 2010. It has also been estimated, for instance, that caries
in ancient times, while humans in turn have won the battle
and periodontal disease are the most costly diseases that the
against microbes in more recent times (e.g., eradication of
majority of the population has to contend with during their
smallpox). Such new diseases are given the terms emerging
lifetime, and the number of working hours lost due to these
infections or re-emerging infections (Fig. 1.1), and they are
infections and the related cost of dental treatment worldwide
broadly categorized as:
amount to billions of dollars per annum (e.g., more than 81
■ new infections: caused by agents such as new influenza
billion dollars in the USA in 2006). This is not surprising as
it is generally accepted that periodontal disease is the most virus strains emerging periodically to cause epidemics,
common affliction of humankind. and the mosquito-borne Zika virus infection.
The advent of the human immunodeficiency virus (HIV) ■ ‘old’ infections: known disease entities where the
infection in the early 1980s and the subsequent concerns on cross aetiological agents have been recently identified through
infection via contaminated blood and instruments have resulted advances in technology (e.g., Helicobacter pylori causing
in an increased regimentation of infection control practices in gastric ulcer disease).
dentistry. Furthermore, many patients are acutely concerned ■ re-emergent infections: diseases that have returned
about possible infection transmission in clinical settings because with a vengeance due to genetic and structural
of the intense, and sometimes unwarranted, publicity given transformations and attendant increased virulence of the
to these matters by the media. The dental practitioner should organism (e.g., drug-resistant Mycobacterium tuberculosis
therefore be conversant with all aspects of infection control in with its ‘new bag of tricks’).
2 1 Why study microbiology?
Diphtheria
Human vCJD
H5N1
monkeypox Hepatitis C influenza
E. coli O157:H7 Lyme disease
Typhoid fever
West Nile virus
Whitewater Anthrax
Arroyo virus Vancomycin-
bioterrorism resistant S.aureus
Dengue
Rift Valley Nipah virus
fever
Hantavirus
Lassa fever HIV
pulmonary syndrome
Human
monkeypox Hendra virus
Plague
Drug-resistant malaria
Enterovirus 71
Ebola haemorrhagic fever
Fig. 1.1 Global prevalence of some emerging and re-emerging diseases. E. coli, Escherichia coli; HIV, human immunodeficiency virus; SARS, severe acute respiratory
syndrome; S. aureus, Staphylococcus aureus; vCJD, variant Creutzfeldt–Jakob disease.
The reasons for their emergence are manifold and include: to the laboratory for identification of the offending agent, the
■ societal events: economic impoverishment (especially in clinical microbiologist utilizes many methods and techniques,
the developing world), war and civil conflicts, as well as as well as a fair amount of thought and contemplation, to
mass population migration. identify the pathogen/s lurking in the clinical sample. In many
■ health care: new medical devices, organ/tissue situations the pathogen may be dead, in which case other,
transplantation, immunosuppression, antibiotic abuse indirect clues via molecular techniques need to be pursued to
and contaminated blood and blood products. incriminate the suspect pathogen. Once an offending pathogen
■ human behaviour: increasing number of sexual partners,
is identified, antimicrobial chemotherapy is the mainstay of
treatment; a description of chemotherapeutic agents and how
injectable drug abuse.
■ environmental changes: deforestation, drought, floods
they are chosen in the laboratory is given in Chapter 7.
The host responds to infection by mounting an immune
and global warming.
response. A highly abbreviated account of basic immunology is
■ microbial adaptation: emergence of new species from
given in Part 2; supplemental reading is essential to augment this
the wild (e.g., HIV), changes in virulence and toxin material, and the reader is referred to the lists of recommended
production and development of drug resistance. texts for this purpose. Immunological nomenclature is complex
and often difficult: a glossary of terms and abbreviations is
therefore provided as an appendix.
About this book Although there are thousands of offending pathogens,
only some are of direct relevance to dental practice and to
This text is divided into six parts in order to highlight the the comprehension of the mechanisms of disease; these are
different features of microbiology related to dentistry, but it described in Part 3. Arguably this section may appear to be
should be noted that such division is artificial and is merely the most daunting part of the book because of the complex
an attempt to simplify the learning process. nomenclature of microbes; hence only the salient bacterial
The first few chapters in Part 1 essentially describe general genera—some of which are more closely related to dental
microbiological features of bacteria and viruses and how they practice (e.g., streptococci) than others (e.g., legionellae)—are
cause human infections (i.e., pathogenesis). Diagnostic micro- outlined. Similarly, the chapters on viruses and fungi are
biology, by which clinical microbiologists ascertain the nature relatively brief, with thumbnail sketches of only the most
of agents causing various infections, is described in Chapter 6. relevant organisms.
The laboratory aspect of this fascinating subject is analogous to The major infections of each organ system are discussed
the work of a crime detection bureau! When a specimen (e.g., in Part 4, with emphasis on those that are most relevant to
pus, urine) from a patient with an infectious disease is sent dentistry. The student is strongly advised to cross-refer to
About this book 3
organisms and their characteristics (described in Part 3) when preceding narrative, should help the reader to assess knowledge
studying this section, as the microbial attributes and the diseases assimilation in key areas.
they cause form a single continuum. Finally, in most chapters the text is arranged under the fol-
Part 5 specifically outlines the microbial interactions in the lowing important features of microbiology, which the student
craniofacial region, in both health and disease. This section must understand in order to deal with infectious diseases:
should be particularly useful for the later years of the dental
curriculum, to reinforce the studies in conservative dentistry,
■ Epidemiology: spread, distribution and prevalence of
periodontics, oral and maxillofacial surgery and oral medicine. infection in the community.
Last but not least, the subject of cross infection and its
■ Pathogenesis: the means by which microbes cause
control in dentistry is encapsulated in Part 6. It provides a disease in humans, an understanding of which is
comprehensive summary of the routine infection control critical for the successful diagnosis and management of
regimens that must be implemented in every dental practice. infections.
The relevance of this information in routine clinical practice ■ Diagnosis: detection of an infection; this depends on the
cannot be overemphasized, and a thorough understanding of collection of the correct specimen in the most appropriate
this material should pay rich dividends in years to come. manner, and subsequent interpretation of the laboratory
As the student will discover, the comprehensive nature of results.
this text has made almost all the materials significant. Thus the ■ Treatment: antibacterial, antifungal or antiviral therapy
reader will be intellectually challenged to learn a new concept combined with supportive therapy leads to resolution of
or terminology in almost every sentence or phrase. In addition, most infections.
an attempt has been made to summarize the information as key ■ Prevention (prophylaxis): immunization is the most
facts, to serve as an aide-mémoire, at the end of each chapter. It useful mode of preventing diseases such as tetanus and
is important, however, that the subject matter is augmented hepatitis B; however, increasing public awareness of
with additional reading, and it is to this end that the list of diseases and their modes of spread significantly helps to
recommended texts is given. The self-assessment quiz at the curb the spread of infections in the community (e.g., HIV
end of each chapter, which may not cover all aspects of the infection).
Further reading Brooks, J. F., Carroll, K. C., Butel, J. S., et al. (Eds.), (2013). The science
of microbiology (Chapter 1). In Jawetz, Melnick & Adelberg’s Medical
microbiology (26th ed., pp. 1–8). New York: McGraw Hill. e-Book.
Beikler, T., & Flemming, T. F. (2011). Oral biofilm-associated diseases:
trends and implications for quality of life, systemic health and Morse, S. S. (1995). Factors in the emergence of infectious diseases.
expenditures. Periodontology 2000, 55, 87–103. Emerging Infectious Diseases, 1, 7–15.
Bonita, R., Beaglehole, R., & Kjellström, T. (2006). Communicable
diseases: epidemiology surveillance and response (Chapter 7). In
Basic epidemiology (2nd ed., pp. 117–132). Geneva: World Health
Organization.
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PA R T 1
General microbiology
The aim of this section is to present (1) the structural features ■ Bacterial structure and taxonomy
of microbes and how they cause disease, and (2) a perspective ■ Bacterial physiology and genetics
of diagnostic laboratory methods to explain the relationship ■ Viruses and prions
between the scientific basis of microbiology and its practical ■ Pathogenesis of microbial disease
application in patient care. Finally, a comprehensive overview ■ Diagnostic microbiology and laboratory methods
of antimicrobial chemotherapy in dentistry is provided, which ■ Antimicrobial chemotherapy
should be supplemented with additional reading due to its
critical relevance to dental care.
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part 1 • General microbiology
2
Bacterial structure and taxonomy
Classification of all living beings including microbes has been Archaea and Bacteria) and eukaryotes (Greek karyon: nucleus).
attempted by many over centuries (Table 2.1). Traditionally, Fungi, protozoa and humans, for instance, are eukaryotic,
though they were all classified into two kingdoms, plants and whereas bacteria are prokaryotic. In prokaryotes, the bacte-
animals, the classification was arbitrary and based on mor- rial genome, or chromosome, is a single, circular molecule of
phological and growth characteristics. With the development double-stranded DNA, lacking a nuclear membrane (smaller,
of novel techniques, the latter classification was expanded to single or multiple circular DNA molecules called plasmids may
include five kingdoms: monera, protista, plantae, fungi and also be present in bacteria), whereas the eukaryotic cell has
animalia. However, the current understanding based on their a true nucleus with multiple chromosomes surrounded by a
genetic relatedness is that all forms of life fall into three domains: nuclear membrane.
Archaea, Bacteria and Eucarya. The main differences among Bacteria comprise the vast majority of human pathogens,
Archaea, Bacteria and Eucarya are listed in Table 2.2. Note whereas archaea appear rarely to cause human disease and
that taken together, Archaea and Bacteria are also known as live in extreme environments (e.g., high temperature or salt
prokaryotes (see later text). concentrations). Archaea received little attention traditionally
Viruses are not included in this classification as they are as they cannot be easily cultured in the laboratory. Interestingly,
unique, acellular, metabolically inert organisms and therefore recent studies using novel techniques such as pyrosequenc-
replicate only within living cells. Other differences between ing have uncovered their presence in the oral cavity. Some
viruses and cellular organisms include: studies have even shown that certain species of archaea are
■ Structure. Cells possess a nucleus or, in the case of more frequently found in subgingival plaque in periodontal
bacteria, a nucleoid with DNA. This is surrounded by the disease.
cytoplasm where energy is generated and proteins are
synthesized. In viruses, the inner core of genetic material
is either DNA or RNA, but they have no cytoplasm and
Morphology
hence depend on the host for their energy and proteins
(i.e., they are metabolically inert).
Shape and size
■ Reproduction. Bacteria reproduce by binary fission (a
The shape of a bacterium is determined by its rigid cell
parent cell divides into two similar cells), but viruses wall. Bacteria are classified by shape into three basic groups
disassemble, produce copies of their nucleic acid and (Fig. 2.1):
proteins, and then reassemble to produce another 1. cocci (spherical)
generation of viruses. As viruses are metabolically inert,
2. bacilli (rod shaped)
they must replicate within host cells. Bacteria, however, can
replicate extracellularly (except rickettsiae and chlamydiae, 3. spirochaetes (helical).
which are bacteria that also require living cells for growth). Some bacteria with variable shapes, appearing as both coccal
and bacillary forms, are called pleomorphic (pleo: many; morphic:
shaped) in appearance.
Eukaryotes and prokaryotes The size of bacteria ranges from about 0.2 to 5 µm. The
smallest bacteria approximate the size of the largest viruses
As mentioned earlier, another modification of classifying (poxviruses), whereas the longest bacilli attain the same length
cellular organisms is to divide them into prokaryotes (i.e., as some yeasts and human red blood cells (7 µm).
8 part 1 2 Bacterial structure and taxonomy
A D G
Ribosomes Cytoplasmic
Capsule membrane
B E H Nucleoid
Fimbriae
C F
Fig. 2.2 A bacterial cell.
Fig. 2.1 Common bacterial forms. (A) Coccus; (B) capsulated diplococci;
(C, D) cocci in chains (e.g., streptococcus) and clusters (e.g., staphylococcus);
(E) bacillus; (F, G) capsulated and flagellated bacillus (e.g., Escherichia coli);
(H) curved bacilli (e.g., Vibrio spp.); (I) spore-bearing bacilli (e.g., Clostridium
tetani); (J) spirochaete.
Arrangement
Bacteria, whichever shape they may be, arrange themselves
(usually according to the plane of successive cell division)
as pairs (diplococci), chains (streptococci), grape-like clusters
(staphylococci) or as angled pairs or palisades (corynebacteria).
Gram-staining characteristics
In clinical microbiology, bacteria can be classified into two
major subgroups according to the staining characteristics of
their cell walls. The stain used, called the Gram stain (first
developed by a Danish physician, Christian Gram), divides
the bacteria into Gram-positive (purple) and Gram-negative
(pink) groups. The Gram-staining property of bacteria is useful
Fig. 2.3 Photomicrograph of a bacterium showing peritrichous flagella. Note the
both for their identification and in the therapy of bacterial relative length of the flagella compared with the size of the organism.
infections because, in general, Gram-positive bacteria are more
susceptible to penicillins than Gram-negative bacteria.
Fimbriae and pili
Structure Fimbriae and pili are fine, hair-like filaments, shorter than
The structure of a typical bacterium is shown in Fig. 2.2. Bacteria flagella, that extend from the cell surface. Pili, found mainly
have a rigid cell wall protecting a fluid protoplast comprising on Gram-negative organisms, are composed of subunits of a
a cytoplasmic membrane and a variety of other components protein, pilin, and mediate the adhesion of bacteria to receptors
(described later). on the human cell surface, a necessary first step in the initiation
of infection. A specialized type of pilus, the sex pilus, forms the
Structures external to the cell wall attachment between the male (donor) and the female (recipient)
bacteria during conjugation, when genes are transferred from
Flagella one bacterium to another.
Flagella are whip-like filaments that act as propellers and guide
the bacteria towards nutritional and other sources (Fig. 2.3). The Glycocalyx (slime layer)
filaments are composed of many subunits of a single protein, The glycocalyx is a polysaccharide coating that covers the outer
flagellin. Flagella may be located at one end (monotrichous, surfaces of many bacteria and allows the bacteria to adhere
a single flagellum; lophotrichous, many flagella) or all over firmly to various structures, for example, oral mucosa, teeth,
the outer surface (peritrichous). Many bacilli (rods) have heart valves and catheters, and contribute to the formation
flagella, but most cocci do not and are therefore non-motile. of biofilms. This is especially true in the case of Streptococcus
Spirochaetes move by using a flagellum-like structure called mutans, a major cariogenic organism, which has the ability to
the axial filament, which wraps around the cell to produce produce vast quantities of extracellular polysaccharide in the
an undulating motion. presence of dietary sugars such as sucrose.
10 part 1 2 Bacterial structure and taxonomy
Capsule
An amorphous, gelatinous layer (usually more substantial than
the glycocalyx) surrounds the entire bacterium; it is composed
of polysaccharide, and sometimes protein (e.g., anthrax bacillus).
The sugar components of the polysaccharide vary in different
bacterial species and frequently determine the serological
type within a species (e.g., 84 different serological types of
Streptococcus pneumoniae can be distinguished by the antigenic
differences of the sugars in the polysaccharide capsule). The
capsule is important because:
■ it mediates the adhesion of bacteria to human tissues or
Cell wall
The cell wall confers rigidity upon the bacterial cell. It is a Pentapeptide N-acetyl
multilayered structure outside the cytoplasmic membrane. bridge muramic acid
It is porous and permeable to substances of low molecular
weight. Fig. 2.4 Chemical structure of cross-linking peptidoglycan component of cell
The inner layer of the cell wall is made of peptidoglycan wall, common to both Gram-positive and Gram-negative bacteria. (After Sharon,
and is covered by an outer membrane that varies in thickness N. (1969). The bacterial cell wall. Scientific American, 220, 92.)
and chemical composition, depending upon the Gram-staining
property of the bacteria (Fig. 2.4). The term ‘peptidoglycan’ is
derived from the peptides and the sugars (glycan) that make
up the molecule. (Synonyms for peptidoglycan are murein
■ The cell walls of some bacteria (e.g., Mycobacterium
and mucopeptide.) tuberculosis) contain lipids called mycolic acids, which
The cell walls of Gram-positive and Gram-negative bacteria cannot be Gram stained, and hence are called acid-fast
have important structural and chemical differences (Fig. 2.5): organisms (i.e., they resist decolourization with acid
alcohol after being stained with carbolfuchsin).
■ The peptidoglycan layer is common to both
Flagella
Pilus
Capsule
(variable)
Outer membrane
Lipoprotein
Peptidoglycan
Periplasmic space
Cytoplasmic
membrane
Cytoplasm
Gram-positive Gram-negative
phospholipid bilayer similar in appearance to that of eukary- composition. They are organized in units of 70S, compared
otic cells. However, eukaryotic membranes contain sterols, with eukaryotic ribosomes of 80S. These differences are the
whereas prokaryotes generally do not (the only exception basis of the selective action of some antibiotics that inhibit
being mycoplasmas). The membrane has the following major bacterial, but not human, protein β-synthesis.
functions:
■ active transport and selective diffusion of molecules and
Cytoplasmic inclusions
solutes in and out of the cell The cytoplasm contains different types of inclusions, which
■ electron transport and oxidative phosphorylation, in serve as sources of stored energy; examples include polymet-
aerobic species aphosphate, polysaccharide and β-hydroxybutyrate.
■ synthesis of cell wall precursors
C Genotypic taxonomy
Keratin coat In contrast to the classical phenotypic classification methods
of spore Peptidoglycan outlined earlier, genotypic classification and speciation of
organisms are becoming increasingly important and useful.
Genotypic taxonomy exploits the genetic characteristics, which
are more stable than the sometimes transient phenotypic features
D of organisms. These methods essentially evaluate the degree
Fig. 2.6 The cycle of sporulation. (A) Vegetative cell; (B) ingrowth of cytoplasmic
of DNA homology of organisms in order to speciate them, for
membrane; (C) developing forespore; (D) forespore completely cut off from the cell example, by assessing molecular guanine and cytosine (GC)
cytoplasm; (E) development of cortex and keratin spore coat; (F) liberation of spore content, ribotyping, random amplification of polymorphic
and conversion to vegetative state under favourable conditions. DNA (RAPD) analysis and pulsed-field gel electrophoresis
(PFGE). Novel bacterial typing methods based on the nucleotide Table 2.3 Hierarchical ranks in classification of organisms (e.g.,
sequences of ribosomal RNA (rRNA) genes have become a Lactobacillus acidophilus)a
robust way of assessing bacterial identity. Further details of
these methods are given in Chapter 3. Taxonomic rank Example
Additionally, recent research indicates that endogenous bacte- Domain Bacteria
rial habitats in humans, including the oral cavity, harbour a flora
that cannot be cultured using routine laboratory techniques. Kingdom Bacteria
These so-called unculturable species comprise both bacteria Phylum Firmicutes
and archaea, mentioned earlier, and can only be detected by
Class Bacilli
molecular techniques or metagenomics (e.g., by direct amplifica-
tion of 16S RNA). The role of these totally new phylotypes of Order Lactobacillales
bacteria in either disease or health awaits clarification. Family Lactobacillaceae
Both the culturable and unculturable organisms (i.e., the
total microbial community) including biomolecules within a Genus Lactobacillus
defined habitat, such as the human body is given the term ‘core Species Lactobacillus acidophilus
microbiome’. The total collection of resident microbes within a
Note: This is the basic classification although more modern and detailed
the core microbiome is termed the microbiota. The analysis of classifications are also available with further subcategorization of the taxonomic
this microbiome has been greatly facilitated by novel techniques ranks.
such as pyrosequencing (a method of DNA sequencing) and
next-generation sequencing (NGS). The data from such studies
have revealed that the oral cavity in health may contain more
than 1 000 different phylotypes (Fig. 2.9; also see Chapter 31)! of the last two ranks, that is, a combination of the generic
name followed by the species name, for example, Streptococcus
salivarius similar to Homo sapiens, note that the species name
How do organisms get their names? does not begin with a capital letter). The name is usually written
Organisms are named according to a hierarchical system, begin- in italics with the generic name abbreviated (e.g., S. salivarius).
ning with the taxonomic rank domain, followed by kingdom, When bacterial names are used adjectivally or collectively, the
phylum, class, order, family, genus and species (Table 2.3). names are not italicized and do not begin with a capital letter
The scientific name of an organism is classically a binomial (e.g., staphylococcal enzymes, lactobacilli).
14 part 1 2 Bacterial structure and taxonomy
Nasal
Oral Human microbiome sample
Skin
Gastrointestinal
Urogenital
Major ecological
areas within the
microbiome Extract DNA
Which organisms are What are the functions of
present? the community?
OTJ1
Bin similar
sequences OTJ2
into OTUs
OTJ3
NCBI
NCBI Compare sequences BLAST
Compare OTUs to Compare sequences to
databases RDP reference genomes to databases
Silva img/hmp
img/hmp m
MG-RAST
Abundance
Abundance
GATTACA
GATTACA
GATTTCA
GATTTCA
Functions
OTU
Identify OTUs in sample and Phylogenetic view of Identify microbial sequences, Identify genes, pathways
relative frequencies community composition variants and polymorphisms and relative frequencies
in sample in sample
Fig. 2.9 A schematic overview of the uses of bioinformatics for functional metagenome analysis. Microbial community contains numerous bacterial and other
species. Once the total DNA has been extracted, the composition of the community is determined by amplifying and sequencing 16S ribosomal RNA (rRNA) gene. Highly
similar sequences are then grouped as operational taxonomic units (OTUs), which are then recognized by comparisons with databases of already recognized organisms.
OTUs are then analyzed to determine the biomolecular and metabolic functions of the community. (Adapted with permission from Elsevier from Morgan, X.C., Segata N.,
Huttenhower C. (2013). Biodiversity and functional genomics in the human microbiome. Trends in Genetics, 29(1), 51–58.)
Key facts
Note: clinically relevant facts and practice points are italicized; • Structures external to the cell wall of bacteria are flagella
key words are in bold. (whip-like filaments), fimbriae or pili (fine, short, hair-like
• The word ‘microorganism’ (microbe) is used to describe an filaments), glycocalyx (slime layer) and capsule.
organism that cannot be seen without the use of a microscope. • Flagella are used for movement, the fimbriae and pili for
• The main groups of microbes are algae, protozoa, fungi, adhesion and the glycocalyx for adhesion, protection and
bacteria and viruses, with progressively decreasing size. biofilm formation.
• All living cells are either prokaryotic (Archaea and Bacteria) • Cell wall peptidoglycan is common to both Gram-positive
or eukaryotic. and Gram-negative bacteria but thicker in the former; it gives
• Prokaryotes such as bacteria are simple cells with no internal rigidity and shape to the organism.
membranes or organelles. • Peptidoglycan comprises long chains of N-acetylmuramic
• Eukaryotes have a nucleus, organelles such as acid and N-acetylglucosamine cross-linked by peptide side
mitochondria and complex internal membranes (e.g., fungi, chains and cross-bridges.
human cells). • Lipopolysaccharides (LPS) are integral components of the
• Bacteria are divided into two major classes according to outer membranes of Gram-negative (but not Gram-positive)
staining characteristics: Gram-positive (purple) and Gram- bacteria; LPS is the endotoxin and therefore Gram-positive
negative (pink). bacteria cannot produce endotoxin.
Taxonomy 15
• Cell walls of some bacteria such as the mycobacteria contain reactions, cultural requirements, biochemical reactions,
lipids (mycolic acids) that are resistant to Gram staining; antigenic structure and DNA composition.
these bacteria are called acid-fast organisms. • The total microbial community including biomolecules within
• Bacterial cytoplasm contains chromosomal nuclear a defined habitat, such as the human body, is called the core
material: nucleoid, ribosomes, inclusions/storage granules. microbiome.
• Spore formation or sporulation is a response to adverse • The total collection of resident microbes within the core
conditions in Bacillus spp. and Clostridium spp. microbiome is termed the microbiota.
• Taxonomy (systematic classification of organisms into
groups) can be performed according to morphology, staining
Further reading Parahitiyawa, N., Scully, C., Leung, W., et al. (2010). Exploring the oral
bacterial flora: current status and future directions. Oral Diseases,
16, 136–145.
Dewhirst, F. E., Chen, T., Izard, I., et al. (2010). The human oral
microbiome. Journal of Bacteriology, 192, 5002–5017. Wade, W. G. (2004). Non-culturable bacteria in complex commensal
populations. Advances in Applied Microbiology, 54, 93–106.
Human Microbiome Project Consortium. (2012). Structure, function and
diversity of the healthy human microbiome. Nature, 486, 2017–2214.
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part 1 • General microbiology
3
Bacterial physiology and genetics
Stationary
Total CO2
cells
Log count
Viable
cells
Log
1 2 3 4 5
Lag Fig. 3.2 Atmospheric requirements of bacteria, as demonstrated in agar shake
cultures. (1) Obligate aerobe; (2) obligate anaerobe; (3) facultative anaerobe; (4)
microaerophile; (5) capnophilic organism (growing in carbon dioxide-enriched
Time atmosphere). See also Table 3.1.
Fig. 3.1 Bacterial growth curve. Lag, lag phase of growth; Log, logarithmic phase
of growth.
Table 3.1 Effect of oxygen on the growth of bacteria
regulatory events can modify the growth rate. Intracellular Degree of oxygenation Term Example
factors include: Oxygen essential Obligate aerobe Pseudomonas
■ end product inhibition: the first enzyme in a metabolic for growth aeruginosa
pathway is inhibited by the end product of that pathway Grows well under Microaerophile Campylobacter
■ catabolite repression: enzyme synthesis is inhibited by
low oxygen fetus
catabolites. concentration (5%)
Extracellular factors that modify bacterial growth are: Grows in the presence or Facultative Streptococcus
■ temperature: the optimum is required for efficient absence of oxygen anaerobea milleri
activity of many bacterial enzymes, although bacteria
Only grows in the Obligate anaerobe Porphyromonas
can grow in a wide range of temperatures. Accordingly,
absence of oxygen gingivalis
bacteria can be classified as: a
Facultative anaerobes may be subgrouped as capnophiles or capnophilic
• mesophiles, which grow well between 25° and 40°C, organisms if they grow well in the presence of 8%–10% carbon dioxide (e.g.,
comprising most medically important bacteria (that Legionella pneumophila).
grow best at body temperature)
• thermophiles, which grow between 55° and 80°C
(Thermus aquaticus, for instance, grows in hot springs
Bacteria can therefore be classified according to their ability to
and its enzymes such as Taq polymerase are therefore
live in an oxygen-replete or an oxygen-free environment (Fig.
heat resistant, a fact exploited by molecular biologists
3.2, Table 3.1). This has important practical implications, as
in the polymerase chain reaction (PCR) (see later text))
clinical specimens must be incubated in the laboratory under
• psychrophiles, which grow at temperatures below appropriate gaseous conditions for the pathogenic bacteria to
20°C. grow. Thus bacteria can be classified as follows:
■ pH: the hydrogen ion concentration of the environment
■ obligate (strict) aerobes, which require oxygen to grow
should be around pH 7.2–7.4 (i.e., physiological pH) for
because their adenosine triphosphate (ATP)-generating
optimal bacterial growth. However, some bacteria (e.g.,
system is dependent on oxygen as the hydrogen acceptor
lactobacilli) have evolved to exploit ecological niches, such
(e.g., M. tuberculosis)
as carious cavities where the pH may be as low as 5.0.
■ facultative anaerobes, which use oxygen to generate
and the second is catalase, which converts hydrogen peroxide Bacterial genetics
to water and oxygen:
Genetics is the study of inheritance and variation. All inherited
2H2O2 → 2H2O + O2 characteristics are encoded in DNA, except in RNA viruses.
Bacterial genetics 19
5’ 3’ 5’ Cytosine Guanine 3’
N
C G
H
H N H O N
A T C
C C C C
T A
N
H C N H N C
N C C N
T A
O H N
G C
H
A T
H
H
Deoxyribose-phosphate CH3 O H N N
backbone C
C C C C
G C N
H C N H N C
C G
N C C N
A T
O H
Thymine Adenine
3’ Deoxyribose-phosphate backbone
5’
T A
Replication
Chromosome replication is an accurate process that ensures oriC
that the progeny cells receive identical copies from the mother
cell. The replication process is initiated at a specific site on
the chromosome (oriC site) where the two DNA strands are
locally denatured. A complex of proteins binds to this site,
opens up the helix and initiates replication. Each strand then Terminus for
replication
serves as a template for a complete round of DNA synthesis,
which occurs in both directions (bidirectional) and on both Fig. 3.4 Bidirectional replication of a circular bacterial chromosome.
strands, creating a replication bubble (Fig. 3.4). The two sites
at which the replication occurs are called the replication forks.
As replication proceeds, the replication forks move around the The main enzyme that mediates DNA replication is DNA-
molecule in opposite directions opening up the DNA strands, dependent DNA polymerase, although a number of others take
synthesizing two new complementary strands until the two part in this process. When errors occur during DNA replication,
replication forks meet at a termination site. Of the four DNA repair mechanisms excise incorrect nucleotide sequences with
strands now available, each daughter cell receives a parental nucleases, replace them with the correct nucleotides and religate
strand and a newly synthesized strand. This process is called the sequence.
semiconservative replication. Such chromosomal replication Bacteria have evolved mechanisms to delete foreign nucleo-
is synchronous with cell division, so that each cell receives a tides from their genomes. Restriction enzymes are mainly used
full complement of DNA from the mother cell. for this purpose, and they cleave double-stranded DNA at specific
20 part 1 3 Bacterial physiology and genetics
Genes Conjugation
The genetic code of bacteria is contained in a series of units This is the mating of two bacteria, during which DNA is
called genes. As the normal bacterial chromosome has only transferred from the donor to the recipient cell (Fig. 3.6A). The
one copy of each gene, bacteria are called haploid organisms mating process is controlled by an F (fertility) plasmid, which
(as opposed to higher organisms, which contain two copies carries the genes for the proteins required for mating, includ-
of the gene and hence are diploid). ing the protein pilin, which forms the sex pilus (conjugation
A gene is a chain of purine and pyrimidine nucleotides. tube). During mating, the pilus of the donor (male) bacterium
The genetic information is coded in triple nucleotide groups carrying the F factor (F+) attaches to a receptor on the surface
or codons. Each codon or triplet nucleotide codes for a specific of the recipient (female) bacterium. The latter is devoid of an
amino acid or a regulatory sequence, for example, start and stop F plasmid (F−). The cells are then brought into direct contact
codons. In this way, the structural genes determine the sequence with each other by ‘reeling in’ of the sex pilus. Then the F factor
of amino acids that form the protein, which is the gene product. DNA is cleaved enzymatically, and one strand is transferred
The genetic material of a typical bacterium (e.g., E. coli) across the bridge into the female cell. The process is completed
comprises a single circular DNA with a molecular weight of by synthesis of the complementary strand to form a double-
about 2 × 109 and composed of approximately 5 × 106 base stranded F plasmid in both the donor and recipient cells. The
pairs, which in turn can code for about 2000 proteins. recipient now becomes an F+ male cell that has the ability
to transmit the plasmid further. The new DNA can integrate
Genetic variation in bacteria into the recipient’s DNA and become a stable component of
its genetic material. Complete transfer of the bacterial DNA
Genetic variation can occur as a result of mutation or gene takes about 100 min.
transfer.
Transduction
Mutation Transduction is a process of DNA transfer by means of a bacterial
A mutation is a change in the base sequence of DNA, as a virus: a bacteriophage (phage). During the replication of the
consequence of which different amino acids are incorporated phage, a piece of bacterial DNA is incorporated, accidentally,
into a protein, resulting in an altered phenotype. Mutations into the phage particle and is carried into the recipient cell
result from three types of molecular change, as follows. at the time of infection (Fig. 3.6B). There are two types of
transduction:
Base substitution 1. Generalized transduction occurs when the phage carries
This occurs during DNA replication when one base is inserted a segment from any part of the bacterial chromosome.
in place of another. When the base substitution results in a This may occur when the bacterial DNA is fragmented
codon that instructs a different amino acid to be inserted, the after phage infection, and pieces of bacterial DNA the
mutation is called a missense mutation; when the base substitu- same size as the phage DNA are incorporated into the
tion generates a termination codon that stops protein synthesis latter.
prematurely, the mutation is called a nonsense mutation. The 2. Specialized transduction occurs when the phage DNA
latter always destroys protein function. that has been already integrated into the bacterial DNA
is excised and carries with it an adjacent part of the
Frame shift mutation bacterial DNA. Phage genes can cause changes in the
A frame shift mutation occurs when one or more base pairs phenotype of the host bacterium; for example, toxin
are added or deleted, which shifts the reading frame on the production in Corynebacterium diphtheriae is controlled by
ribosome and results in the incorporation of the wrong amino a phage gene. This property is lost as soon as the phage
acids ‘downstream’ from the mutation and in the production DNA is lost in succeeding reproductive cycles.
of an inactive protein. Plasmid DNA can also be transferred to another bacterium
by transduction. However, the donated plasmid can function
Insertion independently without recombining with bacterial DNA.
The insertion of additional pieces of DNA (e.g., transposons) or The ability to produce an enzyme that destroys penicillin
an additional base can cause profound changes in the reading (β-lactamase) is mediated by plasmids that are transferred
frames of the DNA and in adjacent genes (Fig. 3.5). between staphylococci by transduction.
Mutations can be induced by chemicals, radiation or viruses.
Transformation
Gene transfer This is the transfer of exogenous bacterial DNA from one cell
to another. It occurs in nature when dying bacteria release their
The transfer of genetic information can occur by: DNA, which is then taken up by recipient cells and recombined
■ conjugation with the recipient cell DNA. This process appears to play an
■ transduction insignificant role in disease (Fig. 3.6C).
Bacterial genetics 21
Normal
DNA base sequence CAT ACT GAG GTT AGT
Transcription
Translation
C deleted
Fig. 3.5 Events that entail mutation: the effect of New base sequence CAG TAC TGA GGT TAG
the deletion and insertion of a single base on the
amino acid sequence (and the quality of the protein New amino acid glu tyr cys gly
thus produced) is shown. sequence
Transposition Plasmids
This occurs when transposable elements (transposons; see
Plasmids are extrachromosomal, double-stranded circular DNA
below) move from one DNA site to another within the genome
molecules within the size range 1–200 MDa. They are capable
of the same organism (e.g., E. coli). The simplest transposable
of replicating independently of the bacterial chromosome (i.e.,
elements, called ‘insertion sequences’, are less than 2 kilobases in
they are replicons). Plasmids occur in both Gram-positive and
length and encode enzymes (transposase) required for ‘jumping’
Gram-negative bacteria, and several different plasmids can
from one site to another (Fig. 3.6D).
often coexist in one cell.
Transmissible plasmids can be transferred from cell to cell
Recombination by conjugation. They contain about 10–12 genes responsible
When the DNA is transferred from the donor to the recipient for synthesis of the sex pilus and for the enzymes required for
cell by one of the aforementioned mechanisms, it is integrated transfer; because of their large size, they are usually present
into the host genome by a process called recombination. There in a few (one to three) copies per cell.
are two types of recombination: Non-transmissible plasmids are small and do not contain the
1. Homologous recombination, in which two pieces of transfer genes. However, they can be mobilized by co-resident
DNA that have extensive homologous regions pair up plasmids that do contain the transfer gene. Many copies (up
and exchange pieces by the processes of breakage and to 60 per cell) of these small plasmids may be present.
reunion.
2. Non-homologous recombination, in which little
Clinical relevance of plasmids
homology is necessary for recombination to occur. A A number of medically important functions of bacteria are
number of different enzymes (e.g., endonucleases, ligases) attributable to plasmids (i.e., are plasmid coded). The plasmid-
are involved in the recombination process. coded bacterial attributes include:
22 part 1 3 Bacterial physiology and genetics
A
Chromosome Pilus
F+ F- F+ F- F+ (x2)
Plasmid
B
DNA
Empty phage fragments
coat
Phage DNA
Phage Daughter
phage
Cell lysis New
bacterium
C
DNA
fragments
Uptake
Natural New
cell lysis bacterium
D
Insertion sequences with
inverted repeats
■ antibiotic resistance (carried by R plasmids) to a new site, it is usually a copy of the transposon that moves,
■ the production of colicins (toxins that are produced by while the original remains in situ (like photocopying). For
many species of enterobacteria and are lethal for other their insertion, transposons do not require extensive homol-
bacteria) ogy between the terminal repeat sequences of the transposon
■ resistance to heavy metals such as mercury (the active (which mediate integration) and the site of insertion in the
component of some antiseptics) and silver, mediated by a recipient DNA.
reductase enzyme Transposons can code for metabolic or drug-resistance
■ pili (fimbriae), which mediate the adherence of bacteria to enzymes and toxins. They may also cause mutations in the
epithelial cells gene into which they insert, or alter the expression of nearby
■ exotoxins, including several enterotoxins. genes.
In contrast to plasmids or bacterial viruses, transposons
cannot replicate independently of the recipient DNA. More
Transposons than one transposon can be located in the DNA; for example,
a plasmid can contain several transposons carrying drug-
Transposons, also called jumping genes, are pieces of DNA that
resistance genes. Thus transposons can jump from:
move readily from one site to another, either within or between
■ the host genomic DNA to a plasmid
the DNAs of bacteria, plasmids and bacteriophages. In this
manner, plasmid genes can become part of the chromosomal ■ one plasmid to another
chemiluminescent marker. The probes carry a single strand of ■ Taq polymerase (a heat-stable enzyme from T. aquaticus
DNA analogous to the pathogen that is sought in the clinical (hence Taq), a bacterium that lives in hot springs)
sample. There are different types of DNA probe: ■ deoxyribonucleoside 5′-triphosphate (dNTP): adenine,
■ Whole DNA probes are derived from chromosomal DNA guanine, cytosine and thymine
and are used to seek organisms where the genome is not ■ primers (with a known DNA sequence).
24 part 1 3 Bacterial physiology and genetics
Pus
Clinical
specimen Paper point
Direction of
migration
Fragments separated into
bands by electrophoresis
3’ 5’
5’ 3’
3’ 5’
5’ 3’
B Denature
Fig. 3.8 The polymerase chain reaction (PCR). dNTP, Size Agarose
deoxyribonucleoside 5′-triphosphate. markers gel
closed system, using either labelled fluorophores or other similar diagnosis of viral, bacterial and fungal and other
labelled probes. Further advantages are the versatility of the diseases. For instance, amplification of viral DNA in
system, enabling (1) analysis of multiple amplicons at specific a patient sample could be made within hours, and
time sequences during a reaction period, (2) semiquantitative sometimes even before the onset of symptoms.
estimation of the yield and (3) multiplex evaluation of the ■ Amplification of RNA. Here, the RNA molecule has to
products (see previous text). The disadvantage is the relatively be first converted to single-strand complementary DNA
expensive technology. (cDNA) with an enzyme called reverse transcriptase (as it
transcribes the RNA code into DNA in a reverse manner).
Why is PCR so widely used? Once this initial step is carried out, the PCR primers and
Taq polymerase are added; afterwards, the experimental
Some reasons why the use of PCR is so widespread: procedure is identical to the standard technique.
■ To study minuscule quantities of DNA, as a single ■ Comparison of different genomes. Random
DNA molecule is adequate for an amplification reaction amplification with short lengths of primers can be used
(hence its use in forensic studies, archaeology and in phylogenetics, the study of evolutionary history and
palaeontology). lines of descent of species or groups of organisms. This
■ Use in rapid clinical diagnostic procedures. The technique is called random amplification of polymorphic
sensitivity of the PCR has resulted in its use in rapid DNA (RAPD).
26 part 1 3 Bacterial physiology and genetics
Subgingival
plaque sample
Culture
Extract DNA directly from sample
and PCR amplify 16S rRNA gene
from all bacteria in the plaque
DNA cut with restriction enzyme Cloned DNA cut with restriction
and fragments separated by gel enzyme and fragments separated by
electrophoresis to obtain fingerprint gel electrophoresis to obtain fingerprints
Compare
fingerprints
Profile 1 2 3 4 3 4 5 1 6 2
Cloned DNA from 16S rRNA genes
which do not match fingerprints from
isolated bacteria (light blue and dark grey profiles).
Compare with 24 000 rRNA gene sequences
in databases to obtain 'identity' of uncultured bacterium
pieces by restriction enzymes and the resultant fragments are ‘global’ perspective of how an organism responds to a specific
separated in an agarose gel with the help of a pulsed electric stress, drug or toxin.
field, in which the polarity is regularly reversed. Large pieces
of chromosomes usually do not separate in conventional
agarose gels, hence the necessity of the pulsed/reversed electric
Proteomics
field. This is defined as the study of the myriad of proteins expressed
by the genome of either an organism, cell or tissue type. Pro-
Pyrosequencing teomics builds on and complements the knowledge gained
from genomics by revealing the levels, activities, regulation
Pyrosequencing is one of the most novel and reliable tech- and interactions of every protein in an organism or a cell. Study
niques of DNA sequencing. It is based on the ‘sequencing of the proteome is more complex than that of the genome
by synthesis’ principle. So called as it relies on the detection as the number of proteins in an organism/cell is considered
of pyrophosphate release on nucleotide incorporation, rather many orders of magnitude greater than that of the number
than chain termination with dideoxynucleotides used in PCR of genes.
techniques. It uses chemiluminescence enzyme reactions and Such complexity is further confounded by the dynamic
photodetection techniques that are highly automated, rapid changes in the proteome in response to the environment and
and sensitive. also the multiple possible interactive combinations among
proteins. Protein chips that can simultaneously identify
Next-generation sequencing large numbers of proteins are helpful in unravelling such
complexity.
Next-generation sequencing (NGS), also known as high-
throughput sequencing, is the catch-all term used to describe
a number of different modern sequencing technologies (e.g., Transcriptomics
Illumina, Ion Torrent). NGS permits sequencing DNA and
This is a related branch of molecular biology that deals with the
RNA much more quickly and cheaply than other previously
study of messenger RNA molecules produced in an individual
developed technologies and has revolutionized microbiology.
or population of a particular cell type.
NGS techniques are now widely used in the analysis of the
oral microbiome.
Metabolomics
The era of ‘-omics’ This is defined as the scientific study of chemical processes
involving metabolites of a cell or an organism. While pro-
With the advent of the new millennium, there has been an teomic analyses do not tell the whole story of what might
explosion of digital and computer technology, the use of which be happening in a cell, metabolic profiling can give an
has led to a parallel advancement of the knowledge of our instantaneous snapshot of the physiology of that organ-
biosphere. This in turn has led to focal developments of sub- ism. This has led to the development of a further domain
disciplines such as genomics, proteomics and metabolomics: known as interactomics. The latter is defined as a discipline
the so-called -omics era. These new technologies have had a involving the intersection of bioinformatics and biology that
significant impact on the identification of microbes, particularly deals with studying both the interactions and the conse-
those that could not be cultured in the laboratory (unculturable quences of those interactions between and among proteins,
bacteria), and on the elucidation of their pathogenic mechanisms and other molecules within an organism. The network of
such as resistance to antibiotics. A brief introduction to the all such interactions is called the interactome. In essence,
various -omics domains are as follows. interactomics aims to compare networks of interactions (i.e.,
interactomes) between and within species in order to elu-
cidate how the traits of such networks are either preserved
Genomics or varied.
This refers to the study of the identity of all genes within One of the current challenges of science is to integrate
the chromosome of a cell. The human animal and micro- proteomic, transcriptomic, metabolic and interactomic data
bial genome sequencing projects have thus far provided a to provide a more complete picture of living organisms.
rich genetic resource to better understand human diseases
including oral diseases. As mentioned in Chapter 2, the
development of technologies such as microarray analysis has
Bioinformatics
helped microbiologists to explore patterns of gene expres- Bioinformatics is an essential component of the -omics era. The
sion in various infectious diseases, and their pathogenic avalanche of information spewed out, for instance, by NGS and
mechanisms, for example, in periodontal disease. The sub- similar techniques cannot be sorted using traditional methods.
category of functional genomics deals with the organization Hence new computational methods and their application to
of the genes and their expression patterns under defined the solution of biological problems, often via the mining of
conditions. information databases, have been developed over the last decade
The development of computer models for high throughput or so. This rapidly developing field is called bioinformatics,
analyses of genomic data has simplified the exploration of and forms a crucial pivotal point for the -omics technology. As
gene expression profiles in both eukaryotes and prokaryotes. bioinformatics occupies a central role in a broad spectrum of
Furthermore, DNA microarray technologies help investigators biological research, its analytical toolkits are equally diverse
evaluate gene expression on a genome-wide basis, providing a and complex.
28 part 1 3 Bacterial physiology and genetics
Key facts
• Bacteria, like all living organisms, require oxygen, hydrogen, • Gene transfer in bacteria may occur by conjugation,
carbon, inorganic ions and organic nutrients for survival. transduction, transformation or transposition.
• Other factors that modify growth are end product inhibition • Plasmids are extrachromosomal, double-stranded circular
and catabolite repression, and the temperature and pH of the DNA molecules capable of independent replication within the
medium. bacterial host.
• Bacteria reproduce by binary fission, leading to logarithmic • The clinical relevance of plasmids lies in the fact that they
growth of cell numbers; the doubling or mean generation time code for antibiotic resistance, resistance to heavy metals,
of bacteria can vary from minutes to hours or days. exotoxin production and pili formation.
• Bacterial growth in laboratory media can be divided into a lag • Transposons are ‘jumping genes’ that move from one site to
phase, log phase, stationary phase and decline phase. another either within or between the DNA molecules.
• Depending on their oxygen requirements, bacteria can be • Gene cloning is the introduction of foreign DNA into another
divided into obligate aerobes, facultative anaerobes, obligate cell where it can replicate and express itself.
anaerobes and microaerophiles. • Gene probes used in diagnostic microbiology are labelled
• Bacterial chromosomes comprise a single, continuous strand (with chemicals or radioactively) pieces of DNA that can be
of DNA with a closed, circular structure attached to the cell used to detect specific sequences of DNA of the pathogen (in
membrane. the clinical sample) by pairing with the complementary bases.
• DNA replication is the synthesis of new strands of DNA using • The polymerase chain reaction (PCR) is a widely used
the original DNA strands as templates. technique that enables multiple copies of a DNA molecule to
• DNA replicates by a process called semiconservative be generated by enzymatic amplification of the target DNA
replication; DNA-dependent DNA polymerase is the main sequence.
enzyme that mediates DNA replication. • Pyrosequencing is a rapid, reliable sequencing method
• Restriction enzymes of bacteria delete foreign nucleotides of relatively short DNA templates based on real-time
from their genomes. These enzymes are therefore extremely (quantitative) pyrophosphate release and is a valuable tool for
useful in molecular biological techniques. identification of bacteria (particularly unculturable).
• Genetic variations in bacteria can occur by either mutation or • Next-generation sequencing (NGS) is a general term
gene transfer. used to describe a number of different modern sequencing
• Mutation, a change in the base sequence of the DNA, can technologies.
be due to either base substitution frame shifts or insertion of
additional pieces of DNA.
Further reading Moat, A. G., Foster, J. W., & Spector, M. P. (2002). Microbial physiology.
New York: Wiley-Liss.
Alberts, B., Johnson, A., Lewis, J., et al. (2007). The molecular biology of Mukherjee, S. (2016). The gene: An intimate history. New York: Scribner.
the cell (5th ed.). New York: Garland.
Beebee, T., & Burke, J. (1992). Gene structure and transcription (2nd ed.).
Oxford: IRL Press/Oxford University Press.
part 1 • General microbiology
4
Viruses and prions
Viruses are one of the smallest forms of microorganism and are visualized electron microscopically, are called peplomers
infect most other forms of life: animals, plants and bacteria. (loosely referred to as ‘spikes’).
They can also cause severe acute oral and orofacial disease,
produce oral signs of systemic infection and be transmitted to Viral nucleic acid
patients and dental staff. The main features that characterize
viruses are: Viral nucleic acid may be either DNA or RNA. The RNA, in turn,
may be ss or ds, and the genome may consist of one or several
■ small size (10–100 nm), averaging about one-tenth the
molecules of nucleic acid. If the genome consists of a single
size of a bacterium molecule, this may be linear or have a circular configuration. The
■ genome consisting of either DNA or RNA but never both;
DNA viruses all have genomes composed of a single molecule
single- (ss) or double-stranded (ds); linear or circular (the of nucleic acid, whereas the genomes of many RNA viruses
encoding of the whole of the genetic information as RNA consist of several different molecules or segments, which are
in RNA viruses is a situation unique in biology) probably loosely linked together in the virion.
■ metabolic inactivity outside the cells of susceptible
of viruses, notably the retroviruses and poxviruses. papova and pox, H for herpes and AD for adenoviruses. Most
of the remainder are RNA viruses, including the self-evident
picornaviruses.)
Taxonomy The following is a concise description of the families of
mammalian viruses.
Vertebrate viruses are classified into families, genera and species.
The attributes used in classification are their symmetry, the
presence or absence of an envelope, nucleic acid composition
DNA viruses
(DNA or RNA), the number of nucleic acid strands and their
polarity. Classification of some of the recognized families of RNA
Papovaviruses
and DNA viruses is given in Table 4.1. (Note: to memorize which Papovaviruses are small, icosahedral DNA viruses with a
viruses contain DNA, remember the acronym ‘PHAD’: P is for capacity to produce tumours in vivo and to transform cultured
Taxonomy 31
Nucleocapsid Nucleocapsid
Capsid
A Naked icosahedral
Capsomeres
Nucleic acid
Spikes/peplomers
(glycoprotein)
D Enveloped helical
Fig. 4.3 Structural components and symmetry of different viruses. (A) Naked icosahedral; (B) naked helical; (C) enveloped icosahedral; (D) enveloped helical.
cell lines. The name ‘papova’ is an acronym derived from the cultured adenoid tissue eliciting cytopathic effects. Syndromes
papillomavirus, polyomavirus and vacuolating agent simian associated with adenoviruses include:
virus 40 (SV40), which make up this family. ■ acute febrile pharyngitis (primarily in infants and
other common disinfectants owing to the disruption of the a poxvirus, as is smallpox, which is now a disease of only
outer lipid envelope. historical interest. Humans occasionally acquire infection by
During reproduction, maturation of the progeny begins in animal poxviruses, for example, cowpox.
the nucleus of the host cell, which buds through the nuclear
membrane and acquires the viral envelope. Typical and highly Parvoviruses
pathognomonic intranuclear inclusions are therefore found
in cells that have undergone active virus replication. As many Parvoviruses are icosahedral viruses with ssDNA. Three
herpesviruses can fuse with the cells they infect, polykaryocytes serologically distinct types of autonomous parvoviruses are
or giant cells readily appear in tissue lesions. Such cells, for recognized in human disease. The first group is found in stool
example, Tzanck cells or nuclear inclusions (Lipschutz bodies), specimens, the second (the B19 virus) in the serum of asymp-
are hallmarks of herpetic infections. tomatic blood donors, whereas the third has been recovered
Different herpesviruses cause a variety of infectious diseases, from synovial tissues of rheumatoid arthritis patients. The B19
some localized and some generalized, often with a vesicular virus is responsible for a febrile illness, particularly in children,
rash. Herpesviruses establish latent infection, which can be manifesting as a maculopapular rash.
readily reactivated by immunosuppression (Table 4.2). The exanthem is characterized by a fiery-red rash on the
The nomenclature of herpesviruses is contentious; there is cheeks: the ‘slapped-cheek’ syndrome (also termed fifth disease).
thus a historical or a traditional (trivial) nomenclature and an
official name for each virus (Table 4.3). The herpesviruses that Hepadnaviruses
commonly infect humans can be distinguished by their antigenic Hepadnaviruses are small, spherical DNA viruses causing
and genomic profiles, although they cannot be differentiated hepatitis, chronic liver infections and possibly liver cancer.
by electron microscopy owing to identical capsid morphology. They are of particular interest in dentistry because of their
They also have a universal ability to establish latent infection mode of transmission via blood and saliva (see Chapter 29).
in the host in which they reside, and manifest a number of
common epidemiological features. Herpes simplex virus, herpes RNA viruses
zoster virus, Epstein–Barr virus, human cytomegalovirus and
herpesviruses 6 and 8 can all cause infections in oral and perioral
tissues (Fig. 4.4); see Chapter 35 for details.
Picornaviruses
Picornaviruses are the smallest family of RNA viruses but
Poxviruses incorporate a very large group of viruses, including the genus
The poxviruses are the largest viruses to infect humans or
animals. Molluscum contagiosum in humans is caused by
Table 4.3 Official and trivial nomenclature of human herpesviruses (family Herpesviridae)
Subfamily species Official name Trivial name Acronym
Alphaherpesvirinae Human herpesvirus 1 Herpes simplex virus 1 HSV-1
Human herpesvirus 2 Herpes simplex virus 2 HSV-2
Human herpesvirus 3 Varicella-zoster virus VZV
Betaherpesvirinae Human herpesvirus 5 Cytomegalovirus HCMV
Human herpesvirus 6 Roseola virus, Herpes lymphotropic virus HHV-6
Gammaherpesvirinae Human herpesvirus 4 Epstein–Barr virus EBV
Human herpesvirus 7 – HHV-7
Human herpesvirus 8 Kaposi’s sarcoma associated herpesvirus HHV-8/KSHV
HCMV, Human cytomegalovirus.
Viral replication 33
7. Release
6. Assembly
Progeny
DNA Late proteins
Host cell
1. Adsorption
+
2. Penetration 3. Uncoating Early proteins
(mainly enzymes)
4. Transcription
Early
mRNA 5. Translation
Fig. 4.5 Steps in the replication of a DNA virus. mRNA, Messenger RNA.
known as haemagglutinin, to certain glycoproteins or and host ribosomes are free to receive viral mRNA and
glycolipids on the host cell). provide a focus for transcription and synthesis of viral
2. Penetration or uptake. The process by which the virus or proteins.
its genome enters the host cell cytoplasm. Penetration can 5. Synthesis of viral components. Viral proteins are of two
be achieved by three separate mechanisms: types:
• endocytosis: most of the virions taken up by • structural (the proteins that make up the virus
endocytosis appear to be degraded by lysosomal particle)
enzymes and therefore fail to initiate infection, but this • non-structural (enzymes required for virus genome
is the normal route to successful infection by many replication).
viruses Structural viral proteins are synthesized on cellular polyri-
• fusion: direct fusion of the viral envelope with the bosomes. There is a simultaneous synthesis of progeny viral
plasma membrane of cells allows the nucleocapsid of nucleic acid, using newly synthesized nucleic acid polymerases.
some viruses to be released directly into the cytoplasm 6. Assembly. Viral assembly is accomplished by
without an intervening phagocytic process incorporation of viral nucleic acid into putative
• translocation: some non-enveloped viruses have capsomeres: procapsids. Assembly may occur in the cell
the capacity to pass directly through the plasma nucleus, cytoplasm or (with enveloped viruses) at the
membrane. plasma membrane.
3. Uncoating and eclipse. After penetration, there is a 7. Release may occur either through gradual budding, in
period during which no intact infectious virus can be the case of enveloped viruses, or by sudden rupture.
detected. This ‘eclipse phase’ begins with uncoating of
The foregoing is a brief, composite picture of processes involved
the lipid membrane and protein capsid surrounding the
in viral multiplication. It should be noted that the replication
nucleic acid viral core. As uncoating proceeds, the viral
cycle of each family of viruses has unique characteristics that
nucleic acid becomes free to act as a template for the
differ from other viruses.
synthesis of virus mRNA.
4. Transcription. The virus mRNA codes for the synthesis of
enzymes necessary to complete the process of uncoating
itself and also to initiate early steps in viral replication. Pathogenesis of viral infections
When the virus initiates the reproductive cycle within
the host cell, the synthesis of host cell RNA is halted, See Chapter 5.
Prions and prion diseases 35
to late life (40–60 years); the clinical course lasts for about
Cellular antiviral response 7–18 months.
That great financial strategist, Mr. Plumley, sat drinking whisky and
water by lamplight. His pipe lay at his side. He had tried to smoke it;
but tobacco flurried him.
“It should be about settled by now,” he muttered. “Where’s that
Bolton?”
“Rap!” came the answer, upon such an acute nervous centre, that
he started as if he had been stung.
He rose, made an effort to compose himself, and went to the door.
A spare tall figure detached itself from the dark, and entered.
“What the devil’s been keeping you?” growled the ex-remover.
“Ah! you’re short-sighted, my friend,” said Mr. Bittern, and walked
coolly into the parlour.
Mr. Plumley stared, felt suddenly wet, shook himself, and followed.
When it came to creeping flesh, he felt the full aggravation of his
size. The slow march of apprehensions, taking time from a sluggish
but persistent brain, seemed minutes encompassing him.
“So,” said the lawyer, dry and wintry, the moment he was in, “you
coveted your neighbour's one ewe lamb?”
Mr. Plumley took up his pipe, blew through it, put it down again,
and said nothing.
“You’d heard of Gardener’s aunt’s little bequest to him of fifty
pounds, duty free, eh?” asked the lawyer.
“No,” said Mr. Plumley.
“O!” said the lawyer. “He bid fifty pounds for that picture of yours
this afternoon, and got it. On whose instructions?”
“Ask him, sir. He acts for many.”
“It wasn’t on yours, then?”
“Is that reasonable, Mr. Bittern, when to my knowledge the man
wasn’t worth a brass farden?”
“What do you say about holding him to his bargain?”
“I say, if he’s bought the picture, he must pay for it.”
“And who bid against him? You don’t know that either, I suppose?”
“Nat’rally. Was I there?”
“Well, I’ve settled for him with Bull and Hacker, and brought you
their cheque, less commission and distraint. Give me a receipt for it.”
The great creature, elated with his own strategy as he was, could
hardly draw it out, his hands shook so. But he managed the business
somehow. The lawyer examined the paper, and buttoning it into his
pocket, took up his hat.
“O, by the way!” he said, as if on an afterthought, “I was forgetting
to mention that Gardener, after securing the picture, put it up to
auction again, at the particular request of some late arrivals, and was
bid a thousand pounds for it. It turned out to be a very good work.”
Mr. Plumley took up his pipe again quite softly, looked at it a
moment, and suddenly dashed it to smithereens on the floor.
“It was a plant!” he cried in a fat, hoarse scream. “I’ll be even with
him—I’ll have the money—the picture was mine—I’ll—by God, I say,
it was a conspiracy!”
The lawyer at the door lashed round on him like whipcord.
“And that’s what I think,” he shouted. “The meanest, dirtiest trick
that was ever played by a canting scoundrel on a poor brother. But I
may get to the bottom of it yet, from the opening scheme to enlist
Gardener’s sympathies for a poor martyr to conscience, to the last
wicked design upon him in the saleroom. I may get to the bottom of
it, cunning as it was planned; and, when I do, let some look out!”
As he flung away, he let in a new-comer, Mr. Bolton, by the
opened door. Mr. Plumley, choking in the backwater of his own fury,
had sunk into a chair, gasping betwixt bitterness and panic. He could
not, for the moment, remember how far he had committed himself.
He looked up to meet the insolent, ironic smile of his confederate.
“Come along, dear boy,” said Mr. Bolton. “Curtain’s down. Cash up!”
He presented a claim for fifty pounds, and stood, his hat cocked
on his head, picking his teeth.
“What’s this curst gammon?” sneered Mr. Plumley, rousing
himself.
“Commission,” said the actor airily. “Five per cent. on the ultimate
selling price of a picture.”
“It went at fifty.”
“Pardon me, sir. Ultimate—ultimate, see agreement” (he smacked
his chest). “One thou’ was the figure, and dirt cheap. Fine example.
I’ll trouble you for a cheque.”
“Two pound ten. I’ll give it you in cash.”
Mr. Bolton whistled a stave, and turned round, his hands deep in
his breeches’ pockets.
“I can sell to the other party. Good day to you, and look out.”
[The End]
TRANSCRIBER’S NOTES
Minor spelling inconsistencies (e.g. cash-box/cash box,
frockcoat/frock-coat, etc.) have been preserved.
Alterations to the text:
[A gallows-bird]
Change “convolutions of war, the merry, the dance-maccabre” to
danse-macabre.
[Our lady of refuge]
“fort of San Fernando. and the cursed French garrison,” change
period to comma.
[The five insides]
(“ ‘Eh, says the old man, ’usky-like, and starting) add right single
quotation mark after Eh.
“a bit forward—‘No, no, no no, no, no, no—’ ” add comma after
third no.
[The jade button]
“The property was recovered—but for the heir…” add period to
sentence.
[End of text]
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