Chapter 1
Molecular Medical Microbiology
The Expanding Concept
Yi-Wei Tang1, Max Sussman2, Dongyou Liu3, Ian R. Poxton4 and Joseph D. Schwartzman5
1
Memorial Sloan-Kettering Cancer Center, New York, NY, USA and Weill Medical College of Cornell University, New York, NY USA, 2Institute of
Cell and Molecular Biosciences, Medical School, Newcastle upon Tyne, UK, 3Royal College of Pathologists of Australasia Quality Assurance
Programs, New South Wales, Australia, 4University of Edinburgh, Edinburgh, UK, 5Geisel School of Medicine at Dartmouth, Hanover, NH, USA
Medical microbiology has long had a molecular
dimension. From the earliest days of microbiology the
phenotypic descriptions of microorganisms included bio-
chemical and physiological details that were in fact
molecular. The word ‘molecular’ seems first to have been
applied to microbiology by Zinsser and Parker in 1923 at
the Columbia University, New York, Department of
Bacteriology. In the course of their work on bacterial
‘hypersusceptibility’, they suspected that they were ‘deal-
ing with substances of lower molecular structure than pro-
teins’ [1]. The term molecular microbiology as it has
more recently come into common use, describes an
approach to the study of microorganisms based to a great
extent on their genetic characteristics. The origins of this
trend, as of much else in modern biology, can be seen in
the seminal description of the structure of DNA by
Watson and Crick [2] and their immediate recognition
that implicit in this structure is the mechanism by which
genetic material is copied. The work of more than half a
century since the structure of DNA was elucidated has
yielded information that has radically changed views
about bacteria and not least those about pathogenic bacte-
ria. The recognition that this new approach has also fun-
damentally changed medical and clinical microbiology
was the inspiration on which the first edition of
Molecular Medical Microbiology [3] was founded in
2002. The object was to provide a general review of the
bacteria of medical importance accessible to practitioners
who need an introduction to the molecular aspects of
medical bacteriology. The developments of the last
decade have meant that a second edition is now desirable.
The development of medical bacteriology since the
pioneering discoveries of the 19th century can broadly
be divided into pre-molecular and molecular phases.
Molecular Medical Microbiology. DOI: http://dx.doi.org/10.1016/B978-0-12-397169-2.00001-9
© 2015 Elsevier Ltd. All rights reserved.
An important account of the state of medical bacteriology
in the late pre-molecular age up to the 1930s is detailed
in A System of Bacteriology in Relation to Medicine [4]
and in William Bulloch’s The History of Bacteriology of
1938 [5]. It is important to recall that at that time virology
and immunology were still regarded as integral parts of
bacteriology; both of these then still embryonic sciences
were to give rise to the major independent sciences of
virology and immunology. The intensive study of the bac-
teriophages, mainly those of Escherichia coli, which
began in the 1940s, would ultimately lead to the impor-
tant concepts and tools of molecular biology.
The molecular approach to the study of microbiology
has had a profound effect on the advance of knowledge,
not only about bacteria in general but also about patho-
genic bacteria in particular; the rate of progress in recent
years has been particularly striking. The complexity of
intact organisms was the great difficulty and weakness
inherent in the analytical study of individual phenotypic
characteristics. This contrasts with the power of methods
by which genes can be cloned and their phenotypes stud-
ied in isolation. It seems remarkable how much was
achieved in the past by the old methods and approaches
that are now seen as slow and laborious. In the case of
pathogens, the most striking advances have been in the
understanding of the mechanisms of bacterial pathogene-
sis [6]. It is particularly fascinating that the mechanisms
of pathogenesis of phenotypically quite different organ-
isms may be similar or identical. Indeed, in some cases,
the molecular genetic evidence suggests that these
systems have passed between bacteria by gene transfer at
some time during the remote or even quite recent past.
The importance of the molecular approach to medical
microbiology goes far further than merely providing
1
2 Molecular Medical Microbiology The Expanding Concept
information about mechanisms of pathogenicity. The cen-
tral objective of medical science is the prevention of dis-
ease. The first great preventive insights, based on
epidemiological observations, were the recognition by the
social pioneers of the 19th century of the importance of
clean drinking water and the safe disposal of sewage. Of
almost equal importance was the discovery of toxoids at
the end of the 19th century. This, together with later
developments in immunology and the manipulation of
genes and immunogens, has given rise to highly success-
ful vaccines. A striking example is the vaccine against
Haemophilus influenzae type b, which promises to elimi-
nate the risk of childhood meningitis due to this virulent
pathogen [7,8]. As will be apparent from the chapters that
follow, molecular biology is yielding far-reaching insights
into every branch of medical microbiology.
A BRIEF HISTORY
A detailed account of the origins of molecular bacteriol-
ogy would lead us too far afield, but a brief sketch of its
development may be helpful. A classical account of the
subject is that by Cairns et al. [9].
The rapid development of the genetics of higher plants
and animals in the early 20th century at first had little
effect on bacteriology and the mechanisms of inheritance
in bacteria were thought to be of quite a different kind.
What was then called bacterial ‘variation’ was recognized
but its genetic basis remained elusive, though it was sus-
pected by some early bacteriologists [10]. Contemporary
views began to emerge in the 1940s, when biochemical
mutants of Neurospora crassa were first isolated [11] and
the unity of genetics in the biological world was recog-
nized. The foundations of bacterial genetics were laid a lit-
tle later with the demonstration that bacteriophage-resistant
mutants appear spontaneously in bacterial cultures [12].
An early step had been the landmark discovery by
Griffith [13] of the in vivo transformation of
Streptococcus pneumoniae in mice. This was the prelude
to the demonstration by Avery and his colleagues [14]
that Griffith’s transforming substance was, in fact, DNA
and it was also the first demonstration of genetic transfer
in bacteria. The resolution of the structure of DNA as a
double helix [2] and the recognition that this structure has
genetic implications [15] led to the deciphering of the
genetic code that determines protein structure. The tran-
scription of the DNA message to messenger RNA and
subsequent translation into the amino acid sequence of
proteins was recognized as sufficiently fundamental that
it became known as a ‘dogma’, though like all dogma it
did not long survive unmodified.
The discovery of the phenomenon of host-controlled
restriction-modification in bacteriophages led to the isola-
tion of restriction endonucleases, which later became
universal tools for the specific manipulation of DNA.
Bertani and Weigle [16] uncovered that most, but not all,
λ bacteriophage particles grown on E. coli K-12 were
unable to multiply in a strain of E. coli that carries the
prophage P1 (E. coli K-12(P1)). The rare λ phage parti-
cles that succeeded to multiply were subsequently able to
propagate at full efficiency in E. coli K-12(P1). This phe-
nomenon was explained by two functions of the prophage
P1: first, that it restricted the propagation of λ phage in
E. coli K-12(P1) and, second, that it modified the few sur-
vivors and so allowed them to propagate in this strain.
Restriction is a general phenomenon due to enzymes
(restriction endonucleases) that destroy ‘foreign DNA’ by
cutting it up. The cutting occurs at sites that depend on
the specificity of the endonuclease present in particular
cells for particular base sequences in DNA. Restriction
may be seen as a kind of defence mechanism against
extraneous genetic material. Modification is due to the
methylation of DNA by methylase enzymes to protect it
from the action of restriction endonucleases. The subject
has been reviewed by Wilson and Murray [17].
The restriction endonucleases of different organisms
vary in their base sequence specificity. Since the sequence
specificity of restriction endonucleases can be determined,
they have become widely used reagents for cutting DNA at
specific sites, to yield manageable lengths of DNA for
detailed study. Similarly, the DNA ligase of E. coli is an
enzyme that rejoins the ‘nicks’ that may from time to time
occur in DNA and also function in DNA replication.
Methods of DNA analysis based on the application of these
enzymes and techniques for gene transfer became the foun-
dation tools of genetic manipulation and molecular genetics.
For details the reader is referred to standard texts on molec-
ular biology, bacterial genetics and genetic manipulation.
A seminal advance was the discovery of the polymer-
ase chain reaction (PCR) by Kary Mullis in 1983 [18]. It
is a means of amplifying a single sequence of DNA to
produce many copies for a variety of purposes. A number
of different PCR techniques have been developed and
these are used for a variety of purposes. These include
diagnosis for clinical purposes in which characteristic
DNA template sequences in a sample are detected by a
known ‘reagent’ sequence. In this way DNA is synthe-
sized, which itself becomes the template for further syn-
thesis and the number of sequences is exponentially
amplified. PCR is also used for cloning in the course of
genetic manipulation.
A key landmark in molecular microbiology was the pub-
lication, in 1995, of the complete genome sequences of
H. influenzae [19] and Mycoplasma genitalium [20], by use
of the sequencing technology developed by Sanger [21]. The
innovative aspect of this work was to sequence random frag-
ments of genomic DNA and to use computers to assemble
the resulting sequences into increasingly larger contiguous
 Chapter | 1 Molecular Medical Microbiology
sequence reads (so-called ‘contigs’). Microbial genome
sequencing, which originally cost greatly in excess of
$1 million per genome and which could take years to com-
plete, can now be done in a few days for a fraction of the
cost by means of next-generation sequencing techniques
[22]. The rapid progress in this field is such that it is pre-
dicted that genome sequencing will become routine in diag-
nostic microbiology laboratories. This will provide clinicians
with unprecedented information about a given infective
microorganism, including its precise identification, antibiotic
resistance profile, toxin production and other potential viru-
lence factors that may affect therapy and thereby treatment
outcomes [23]. Already, multiple whole-genome sequences
are available for the majority of known human microbial
pathogens and the number of sequences is currently increas-
ing exponentially.
The low cost and ease with which the genomes of
microbial strains can be sequenced has opened up tech-
nology platforms for the analysis of microorganisms in
ways that were unimaginable even a few years ago. For
example, the comparative analysis of the genomes of
related organisms provides insights into their evolution
and particularly their acquisition of genes and groups of
genes, such as antibiotic resistance genes and pathogenic-
ity islands, by horizontal transfer. For example, in a
ground-breaking study, the ancient genome sequence of
Yersinia pestis, responsible for the Black Death that rav-
aged Europe in the Middle Ages, was reconstructed after
the sequencing of DNA extracted from the teeth of vic-
tims interred in a burial ground in East Smithfield,
London [24]. Phylogenetic analysis indicated this ancient
strain is an ancestor of most extant strains of Y. pestis.
Unexpectedly, its perceived enhanced virulence was
found not to be due to phenotypic differences, but proba-
bly the result of prevailing social and environmental
factors. In a more contemporary analysis of the genomes
of 151 strains of Clostridium difficile, it has been possible
to monitor retrospectively the emergence and global
spread of the epidemic strain C. difficile 027/BI/NAP1
and show the existence of two epidemic lineages and a
relationship with rare fluoroquinolone resistance [25].
The availability of whole-genome sequences has
opened up new technologies to study the behaviour of
microorganisms, the so-called ‘omics’ technologies [26].
While the genomes of microorganisms are rather static,
their transcriptomes and proteomes are highly dynamic
and subject to continuous change and adjustment in
response to the environment and phases of the cell cycle.
The use of DNA arrays and more recently RNA-seq tech-
nologies make it possible to obtain snapshots of the gene
expression profile simultaneously in both pathogen and
host [27]. Similarly, two-dimensional gel electrophoresis
and multidimensional liquid chromatography, coupled to
mass spectrometry, facilitate a similar view of the protein
The Expanding Concept 3
composition of the cell under different growth conditions
[28]. Such studies can be extended to study the concentra-
tions of metabolites (metabolomics) and the dynamic flux
of metabolites through the metabolic pathways (fluxo-
mics) [29]. Finally, next-generation sequencing has facili-
tated the new field of metagenomics, with major studies
aimed at cataloguing the microbial flora of, for example,
the human gut, skin and oral cavity, with the aim of better
understanding the complex relationship between humans
and microbes [30].
It will be evident that the large body of molecular
information that is progressively becoming accessible will
explain many hitherto mysterious characteristics of patho-
genic bacteria and their interaction with their hosts. It
should also be clear that deductions from molecular biol-
ogy alone are not sufficient to explain many features of
microbial behaviour; a complete understanding of this
will ultimately depend on the reintegration into ‘whole
organism’ biology of the information obtained by molecu-
lar biological analysis.
MOLECULAR MEDICAL MICROBIOLOGY
In the past much of the practice and thinking in medical
and particularly in clinical bacteriology was based on the
classical views of bacteria and their behaviour. These
views have changed rapidly under the influence of accu-
mulating fundamental molecular knowledge. These
changes, already evident in the better understanding of
bacterial pathogenicity and pathogenesis, are extending
into every branch of medical and clinical microbiology,
from bacterial morphology at one extreme to ecology,
epidemiology and prevention at the other. At the same
time molecular techniques are finding an increasing use
in the diagnostic laboratory. In addition, the molecular
approach has far-reaching implications for the understand-
ing of the interaction between host and microbe [31,32].
This book is an attempt to collect a wide range of
molecular information about all aspects of bacterial
pathogens. Its general structure is, therefore, not unlike
that of the classical textbooks; the more fundamental
aspects of microbiology are considered in the early chap-
ters of the book, before individual pathogens are dis-
cussed in detail. The chapters differ from conventional
texts of medical microbiology in that it is assumed that
information of a more traditional and classical ‘pheno-
typic bacteriology’-kind can be found in many well-
known and excellent texts. This has made it possible to
concentrate on the more recent molecular information that
is increasingly rapidly becoming available.
Medical microbiology, whether molecular or classical,
is now frequently practiced and researched by scientists
rather than by medical practitioners. A number of chap-
ters described as ‘clinical overviews’ have, therefore,
4 Molecular Medical Microbiology The Expanding Concept
been included in the belief that medical microbiology
cannot be seen in its full perspective without reference to
the effects that pathogenic bacteria have on the host.
The new molecular knowledge can be expected increas-
ingly to influence medical bacteriology from its diagnostic
beginning to its therapeutic aim by increasing the speed of
the former and the range and specificity of the latter. It will
also play an increasing part in reaching epidemiological
and preventive objectives. It is interesting to contemplate
the possibility that in future the targets of therapy may
move from structural components of the bacterial cell to the
mechanisms of pathogenesis that are the ultimate causes of
the disease syndromes as observed clinically. A first step in
this direction, at the beginning of the 20th century, was the
discovery of the toxins of Gram-positive organisms and
their neutralizing antitoxins. If, as a result of molecular
insights, the same were possible, for example, for the secre-
tory delivery systems of Gram-negative bacteria, might
some of the problems of antibiotic resistance be circum-
vented? Such possibilities would not only cause excitement
in the research laboratory, but they would also dramatically
change practices and protocols in the diagnostic laboratory.
What seems certain is that the molecular approach will
increasingly change and enhance the understanding and
practice of medical microbiology.
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