Table Of ContentMolecular Methods for
Microbial
Identification and
Typing
K. J. Towner and A. Cockayne
Department of Microbiology and PHLS Laboratory, University Hospital,
Queen's Medical Centre, Nottingham, UK
laDI
SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.
First edition 1993
© 1993 K.J. Towner andA. Cockayne
OriginaUy published by Chapman & HaU in 1993
Phototypeset in 10/12pt Palatino by Intype, London
ISBN 978-0-412-49390-4 ISBN 978-94-011-1506-3 (eBook)
DOI 10.1007/978-94-011-1506-3
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Contents
Preface
v
1 An introduction to microbial identification and typing methods 1
1.1 A brief overview of microbial identification 1
1.2 Conventional biological methods for microbial typing 3
1.3 Molecular approaches to microbial identification and typing 10
References 18
2 Analysis of nucleic acid profiles 28
2.1 Introduction 28
2.2 Plasmid analysis 29
2.3 Analysis of chromosomal DNA 42
2.4 Summary 52
References 53
3 Identification and typing by nucleic acid hybridization techniques
64
3.1 Introduction 64
3.2 Basic principles of nucleic acid hybridization 65
3.3 Analysis of RFLPs by hybridization techniques 76
3.4 Summary 82
References 85
4 Nucleic acid amplification and sequencing techniques 93
4.1 Introduction 93
4.2 The polymerase chain reaction (PCR) 94
4.3 Other amplification methods 105
4.4 Nucleic acid sequencing techniques 108
4.5 Summary 111
References 112
Contents iv
5 Analysis of protein and lipopolysaccharide profiles 123
5.1 Introduction to protein fingerprinting 123
5.2 Protein analysis 124
5.3 Lipopolysaccharide typing schemes 145
5.4 Summary 149
References 150
6 Typing and identification of microorganisms with antibodies 159
6.1 Introduction 159
6.2 Production of antibodies for serotyping and identification 160
6.3 Techniques used in serotyping 165
6.4 Summary 179
References 180
7 Final summary and future prospects 187
Index 194
Preface
This book is intended as a source book for postgraduate students,
research workers and diagnostic microbiologists in all fields of micro
biology, but particularly those active in medical microbiology, who wish
to explore the possibilities of modern molecular methods for microbial
identification and typing.
Many examples of such applications are concealed in the scientific
literature, but rather than dwell in depth on specific organisms or
epidemiological situations, we have tried to introduce the principles of
each individual method and give an overview of the methodology,
advantages and disadvantages implicit in each technique. Thus it is
not a 'recipe' book, but is intended as a first point of reference for
microbiologists seeking an introduction to these techniques.
The development of these methods has resulted in an exciting period
for microbial identification and typing. While many of the methods
have been developed with bacteria in mind, we hope that the lists of
organisms and extensive references to detailed methodology will enable
the interested reader to find an advanced molecular technique that can
be applied readily to their own 'favourite' organism, be it a bacterium,
virus, fungus or protozoan.
It is a pleasure to acknowledge the encouragement received from
friends and colleagues in our own department and elsewhere. We
would particularly like to thank Debbie Cooke and Sarinder Day for
their help with the illustrations. This book would not have been possible
without the tolerance and sustenance provided by Janet Towner and
Fiona MacDonald.
Kevin Towner and Alan Cockayne
Nottingham
An introduction to
microbial
identification and
1
typing methods
1.1 A BRIEF OVERVIEW OF MICROBIAL IDENTIFICATION
Early classification and identification methods for microbes relied on
tests that were based on the phenotypic properties of individual organ
isms. Strains were assigned to somewhat intuitive classification groups
on the basis of small numbers of characteristics which could be exam
ined easily, such as staining properties, morphology, motility,
nutritional requirements, acid production, pigmentation and spore for
mation. Such an approach suffers from the basic drawback that tests
derived for one group of organisms are not always useful for other
groups. Thus, for example, many tests used for identifying different
members of the Enterobacteriaceae are not suitable for identification of
strictly aerobic Gram-negative bacteria. In addition, the introduction of
increasingly objective modern taxonomic methods has led to recognition
of the fact that over-dependence on a small number of properties for
classification purposes can lead to serious errors; indeed, while some
taxonomic groupings based on phenotypic properties have stood the
test of time, other groups have been shown to contain a wide range of
heterogeneous organisms.
Awareness of the limitations of traditional taxonomic methods has
resulted in a re-evaluation of the whole process of microbial identifi
cation and classification. What began as a rather subjective and intuitive
subject has changed radically in recent years. Increasing use of the
molecular identification and typing methods which form the main sub
ject of this book has led to revision of some accepted classification
schemes and the recognition of new relationships among microbes.
2 An introduction to microbial identification and typing methods
However, whatever the criteria for taxonomic groupings, the basic unit
for the purposes of microbial identification remains the species.
Traditionally, a microbial species has been considered to be a group
of strains which show a high degree of similarity in terms of their
phenotypic properties. As indicated above, this is a rather subjective
grouping (a taxospecies) which may be influenced greatly by the ideas
and aims of the taxonomist. Alternative methods of grouping include
the genomic species, a group of organisms with high DNA-DNA hom
ology values, and the genospecies, a group of organisms capable of
genetic exchange.
In recent years it has become accepted by microbial taxonomists that
nucleic acid hybridization and sequencing studies provide the best avail
able and most rational methods for designating species and determining
relationships between different organisms. A complete DNA sequence
forms the reference standard to determine phylogeny, and phylogeny
should determine taxonomy. A formal molecular definition of a species
has been suggested (Wayne et al., 1987), in that a species should,
generally, include strains with approximately 70% or greater
DNA-DNA relatedness, and with SoC or less divergence values (6 Tm);
however, only species which can be differentiated by phenotypic
properties should be given a formal name. Murray et al. (1990) have
pointed out that it is completely impracticable to define genera and
species solely on the basis of phylogenetic data, and that initial charac
terization of genera needs to be on the basis of phenotypic properties.
Nevertheless, these authors recommended that existing classifications
should be re-examined on a molecular basis. The techniques used to
determine such molecular relationships are described in detail in later
chapters of this book. Obviously, while it is desirable to have as few
changes as possible to existing taxonomic arrangements, it is important
that molecular relationships should be reflected accurately in nomencla
ture and classification schemes.
Once individual species have been defined, they can then be arranged
into genera and families, on the basis of overall similarity, to form a
hierarchical system. However, although identification to the species
level is the primary purpose of all microbial classification schemes, the
separation and accurate recognition of subtypes within a species is
assuming greater importance in all branches of microbiology, and par
ticularly in medical microbiology. Indeed, in many cases, control of
communicable diseases would not be possible without the use of typing
methods to help define the sources of infection, mechanisms of trans
mission and rate of spread of infection in a susceptible population.
Other areas in which accurate microbial typing is important include
ecological studies involving the release and monitoring of novel micro
organisms in new natural habitats (Jain et al., 1988), and industrial
Conventional biological methods for microbial typing 3
screening programmes searching for new microbial products (Goodfel
low and O'Donnell, 1989).
As indicated earlier, a complete DNA sequence would form the ulti
mate reference standard for recognizing sub-types within a species.
Short of achieving this ideal, any typing technique relies on finding
detectable differences between isolates. There is a tendency to assume
that organisms which are indistinguishable by a chosen typing method
are identical and have a common source. However, it should always be
remembered that such organisms may be indistinguishable only when
'viewed' through the typing 'window' generated by a particular typing
scheme, and may be totally different when typed by an alternative
method. Neither typing system generates 'wrong' results, but each
provides a different way or angle of looking at the same problem.
As with identification to the species level, many well-established
microbial typing schemes, particularly in clinical microbiology, are
based on the recognition of particular phenotypic properties. While
this book is concerned primarily with modern molecular methods for
microbial identification and typing, it is important that the newer
methods should be considered in comparison with the older established
methods which they may eventually supersede.
1.2 CONVENTIONAL BIOLOGICAL METHODS FOR MICROBIAL
TYPING
Conventional biological typing methods for microorganisms were
developed originally in response to a growing awareness of the different
factors which influence the spread of human pathogens in various
environments. An ideal typing system (Hawkey, 1989) should: (a) be
able to type the vast majority of strains encountered; (b) have good
discrimination with the ability to recognize a reasonable number of
types; (c) show good reproducibility over a long period of time and in
different centres; (d) be readily applicable to natural isolates as opposed
to laboratory collections of strains; (e) should not be too complicated or
expensive. As indicated by the examples in Tables 1.1-1.4, the main
biological typing techniques of biotyping, phage typing, serotyping and
bacteriocin typing are well established and have been applied to a wide
range of microorganisms. Details of the various technical modifications
required for each individual genus can be found in the references indi
cated; however, certain advantages and disadvantages are linked
directly to each of the main typing methods, and these are described
briefly in the following sections.
4 An introduction to microbial identification and typing methods
1.2.1 BIOTYPING
Frequently, initial differentiation within a newly-delineated species is
achieved by examining the cultural and biochemical characteristics of a
large collection of individual strains belonging to the species. Such
characteristics may include colonial morphology, growth requirements,
fermentation ability, carbon source utilization and antibiotic resistances.
In theory such properties are easy to determine, but in practice the
determination of biotypes may not always be straightforward. Differ
ences in colonial morphology are often extremely subtle and, therefore,
can be rather subjective. Biochemical reactions are more dependable,
but may also be difficult to interpret. Many laboratories use commer
cially available galleries of tests, such as the API 20E system with 20
different tests, to provide a biochemical profile from which different
biotypes can be identified. However, apart from instability problems
resulting from occasional loss of plasmids encoding certain metabolic
functions, variations in the duration of incubation and the inoculum
size may affect interpretation of the results, while strains that are freshly
isolated may exhibit different reactions compared with strains that have
been stored. Newer automated biochemical fingerprinting systems,
based on an evaluation of the kinetics of biochemical reactions per
formed in microtitre plates, have been reported to give good reproduc
ibility and discrimination for certain species, such as Enterobacter cloacae
(Kuhn, Tullis and Burman, 1991), but in many cases there may be only
a very limited number of biochemical types within a species, resulting
partly from the fact that traditional taxonomic procedures, particularly
in clinical microbiology, may have relied on a single specific biochemical
reaction to define the species in the first place.
Resistance to a range of antibiotics (the antibiogram), while normally
easy to determine, is often associated with potentially unstable extra
chromosomal R plasmids, and therefore may be subject to frequent and
abrupt multiple changes. The relatively small number of agents with
the potential to give different results also means that discrimination is
poor. Thus, while it is useful to identify recurring resistance patterns
in a local situation, such patterns have little value when considered in
isolation in comparative studies involving several different centres.
In general, it can be concluded that the determination of biotypes
within a species, while possibly helpful in a limited short-term local
investigation, will be useful only in association with other typing
methods for long-term investigations covering different epidemiological
outbreaks or ecological situations. Examples of organisms in which
biotyping schemes have proved useful for epidemiological purposes are
listed in Table 1.1.
Conventional biological methods for microbial typing 5
Table 1.1 Examples of microorganisms in which biotyping schemes have proved
useful for epidemiological purposes
Organism Reference
Acinetobacter spp. Bouvet (1991)
Aspergillus fumigatus Fanti et a/. (1989)
Bacillus spp. Berkeley et al. (1984)
Brucella spp. McCullough (1978)
Campylobacter spp. Kapperud et al. (1984)
Citrobacter spp. Lanyi (1984)
Neisseria gonorrhoeae Catlin (1978)
Pasteurella haemolytica Biberslein (1978)
Salmonella spp. Katouli et a/. (1992)
Serratia liquefaciens Traub (1991)
Streptococcus faecalis Smyth et a/. (1987)
Vibrio cholerae Mukerjee (1978)
1.2.2 PHAGE TYPING
Bacteriophages (phages) are viruses capable of infecting bacteria, lead
ing in some cases to lysis of the bacterial cell and release of further
infective phage particles, while in other cases the phage can persist as
a relatively stable prophage within the bacterial cell. Phage typing is a
method for bacterial strain identification that is based upon sensitivity
to defined collections of bacteriophages which have been selected to
provide the maximum sensitivity for differentiating strains within a
particular species. Typing may be 'direct', i.e. based on direct sensitivity
to either unadapted or adapted phages, or 'indirect', i.e. based on
detection and identification of phages present as prophages in bacteria.
Phage typing schemes are highly sensitive and have been developed
for numerous bacterial genera (Table 1.2), many of which have not yet
been typed successfully by other means. Such typing schemes are used
widely for epidemiological purposes throughout the world. Phage
typing remains the major typing method for Staphylococcus aureus, and
is also particularly useful for subdividing serotypes of Pseudomonas aeru
ginosa and Salmonella/Shigella spp. Nevertheless, the methodology has a
number of important disadvantages and limitations (Meitert and Meit
ert, 1978). Since environmental conditions can affect the sensitivity of
bacteria to infection by phages, phage typing is a technically complex
method in which many variables must be controlled. Some species may
contain too few phage types, while others may contain too many
phage types represented by only one strain. In other cases, e.g., Acineto
bacter (Bouvet, 1991), a phage typing scheme may be reliable with strains
from one country, but may be less effective, with a high proportion of
non-typable strains, with isolates from other parts of the world.
