6       Molecular subtyping of bacteria

 

Infectious disease surveillance of pathogens of importance in veterinary medicine employs two strategies to detect cases and outbreaks, laboratory-based and syndrome-based surveillance. Of these, the former strategy is more accurate since definitive diagnosis of an infection requires laboratory confirmation. A range of methods is available in veterinary diagnostic laboratories, including conventional microbiological methods using traditional culture-based techniques, immunoassays to detect antigens associated with a pathogen of interest, and modern techniques that include analysis of bacterial nucleic acid. The emergence of novel bacteria challenges surveillance efforts and thus these protocols must constantly evolve to ensure the capability of identifying such pathogens. More than 60% of all emerging pathogens are zoonotic in origin.

Characterization of a veterinary pathogen is essential for the support of epidemiological investigations of a disease outbreak. Laboratory methods used must be capable of identifying those organisms linked to the outbreak while excluding isolates unrelated to the outbreak. Conventional laboratory approaches are summarized in the illustration.

Although some phenotype-based methods have been used successfully, many are not universally applicable and mutation or changes in gene complement can result in altered phenotypic expression, leading to incorrect bacterial identification. Accordingly, these former methods limit the reliability of phenotype-based identification for surveillance purposes.

The rapid development in molecular-based approaches has led to the design of new diagnostic protocols that are independent of the inherent limitations of traditional methods. Molecular subtyping methods target variation within the genomes of bacteria and also decrease the limitations encountered with more conventional phenotyping approaches.

Bacterial subtyping methods permit the identification of a bacterium below the species level and, in addition, provide methods for tracking an organism, describing its molecular epidemiology and defining its transmission routes. This modern analytical approach provides a more refined identification of a bacterium, based on its DNA fingerprint, than phenotype-based methods. Compared with traditional diagnostic methods, this facilitates the recognition of different Escherichia coli O157:H7 isolates and Salmonella Typhimurium subtypes among others. Transmission routes of methicillin-resistant Staphylococcus aureus (MRSA) between humans and animals have also been identified by subtyping isolates and comparing their DNA fingerprint patterns. In general, technical approaches to molecular subtyping include the use of restriction fragment length polymorphism (RFLP) analysis, PCR-based amplification of conserved repetitive sequences in bacterial genomes and whole bacterial genome DNA sequencing (Table 6.1).

Table 6.1 Molecular-based subtyping methods used for tracing bacterial pathogens associated with disease outbreaks in animal and human populations.

Sequential development of analytical methods	Molecular basis of subtyping methods
First-generation methods	Plasmid DNA profiling Restriction digestion of purified plasmids
Second-generation methods	Restriction endonuclease digestion of total DNA (including chromosomal and plasmid) Ribotyping
Third-generation methods	Pulsed-field gel electrophoresis (PFGE) PCR-based amplification Rapid amplification of polymorphic DNA (RAPD) PCR-RFLP analysis of conserved genes (flaA, recN and others) PCR ribotyping REP-PCR ERIC-PCR BOX-PCR AFLP
Fourth-generation methods	Multi-locus variable-number tandem repeat analysis (MLVA) Multi-locus sequence typing (MLST) Whole genome-based DNA sequencing

Plasmid profiling, which can also be used to type organisms, involves the purification of plasmids from a bacterium of veterinary importance, followed by their separation on an agarose gel. Notwithstanding some limitations, plasmid profiling is important when characterizing genes encoding antibiotic resistance.

Comparative genomic hybridization

DNA microarrays (see Chapter 4) can also be used for subtyping purposes. Probes attached to the solid surface can detect complementary sequences in bacterial isolates of interest. DNA is purified from a bacterium and labelled either chemically or enzymatically, before being hybridized to the array. Unbound DNA is washed off and signals from hybridized probes are subsequently detected automatically by a scanner. These data are then analysed using suitable software. Together with other molecular methods, DNA microarrays are appropriate procedures for bacterial subtyping.

Commercial DNA microarrays are available and can be used for diagnostic testing and disease investigations. Similarly, the serotypes of E. coli and Salmonella species can now be identified using commercially available DNA microarrays.

Restriction fragment length polymorphism analysis

RFLP analysis requires the purification of the bacterial chromosome and associated plasmid(s), prior to enzymatic digestion with a restriction endonuclease. Electrophoresis produces a multi-band pattern or RFLP pattern in an agarose gel. The RFLP pattern produced is often too complex to serve as a fingerprint and is difficult to analyse, limiting the application of this subtyping method. Moreover, plasmids present initially in a strain may be lost later, altering the banding profile and complicating isolate comparison.

A high-resolution RFLP-based strategy known as optical mapping has been described. Following the gentle lysis of a bacterial cell, this technique facilitates the creation of a high resolution ordered genome map. Purified DNA is stretched out in a microfluidic chamber and digested with a restriction endonuclease enzyme. The resulting DNA fragments remain attached in the chamber in the same order as they appear in the genome. After staining with a DNA intercalating dye to allow visualization by fluorescent microscopy, the lengths of the DNA fragments are measured by fluorescent intensity. Using specialized software, the optical map is subsequently resolved.

Ribosomal-encoding DNA genes (rRNA) are naturally amplified in bacteria and have been used successfully as a target for identification. Large portions of these genes (the rrs genes code for 16S rRNA and the rrl genes code for 23S rRNA) have been conserved throughout evolution. In this procedure, chromosomal DNA is purified and digested with a suitable restriction enzyme, then Southern blotted, before hybridization with a species-specific rRNA probe. The pattern of fragments detected is referred to as the ribotype. Since these genes are highly conserved, pathogens can be identified using appropriately labelled 16S and 23S rRNA probes.

A major limitation with restriction enzyme-based methods is the complexity of the fragmentation patterns generated which renders their analysis difficult to interpret. Pulsed-field gel electrophoresis (PFGE) can overcome this limitation by taking advantage of the digestion of the bacterial chromosome and any associated plasmids using rare cutting restriction endonucleases, also known as macrorestriction analysis. These enzymes cut chromosomal DNA into a lower number of large DNA fragments which can be resolved using specialized electrophoresis equipment. PFGE is regarded as the gold standard in molecular subtyping.

PFGE is a highly discriminating subtyping protocol and is used to determine the genetic relationships between case-related and -unrelated isolates. As the method is relatively simple to perform, standardizing the technical elements facilitates the comparison of PFGE profiles between laboratories nationally and internationally. PulseNet (www.cdc.gov/pulsenet) is an example of a globally standardized and operated PFGE-based subtyping network used to track food-borne pathogens across countries and continents.

PCR-based subtyping methods

Several PCR-based subtyping methods have been developed. In general, these methods are simple to carry out and can be applied to any microbial genome. Some of these approaches are shown in Table 6.1. A brief outline of three methods is given below.

Rapid amplification of polymorphic DNA (RAPD) also known as arbitrarily primed-PCR (AP-PCR) was one of the first examples of PCR-based subtyping described. This method, which does not require any prior knowledge of the organism's DNA sequence, uses a randomly selected primer, along with a low-temperature annealing step during amplification to produce a DNA fingerprint pattern of a bacterium of particular interest.

PCR-RFLP can be applied to a gene target that exhibits a high degree of polymorphism and therefore can be used to discriminate between bacterial isolates. An example of this is flaA subtyping used to subtype Campylobacter jejuni isolates. In this example, the gene of interest, the flagellin A subunit encoding flaA, is amplified by PCR. The amplified PCR product is then subjected to digestion using a suitable restriction endonuclease, in this instance Hinf1, producing an RFLP profile. The RFLP profile is then used to compare different isolates of the same bacterium. Other targets used include 16S, 23S rRNA and the interspacial region 16–23S rRNA, fliC for E. coli O157 and the coa-encoding coagulase gene in Staphylococcus aureus.

Bacterial genomes contain several examples of repetitive sequences along their length. Examples of common repeats include the 38-bp repetitive extragenic palindromic sequence (REP), the 126-bp enterobacterial repetitive intergenic consensus (ERIC) and the 158-bp BOX repeat sequences (Table 6.1). REP-PCR utilizes the nucleotide sequences conserved within the repeat element to facilitate the design of primers located towards the extremities of these repeats and which amplify the DNA regions located between the repeats.

Amplification fragment length polymorphism (AFLP) does not require any prior knowledge of the target bacterial genome sequence. Bacterial genomic DNA is first digested with one or more restriction endonucleases to which synthetic short oligonucleotide adaptors of known sequence are attached using the cohesive ends generated by the restriction enzyme. This forms the sites to which adaptor-specific DNA primers are annealed and used to amplify the adaptor-ligated DNA fragment. A complex pattern of DNA fragments is produced following PCR, ranging in size from 50 to 100 bp with between 40 and 200 bands that are resolved by conventional agarose gel electrophoresis. DNA fragment profiles can be arranged as described earlier and compared with other bacterial isolates.

DNA sequence-based subtyping methods

DNA sequence-based subtyping has emerged as a new approach to distinguish isolates of the same bacterial species. Molecular subtyping methods based on a selected number of genes or the complete genome of a bacterium have been developed. Using purified DNA from their genomes, these methods are being utilized for identification of pathogenic bacteria in animals and humans (Table 6.1). Two of these methods are briefly described.

Multi-locus sequence typing (MLST) is an example of an approach whereby chromosomal DNA is purified and short segments of up to seven housekeeping genes are amplified prior to sequencing. The genes used for MLST analysis all encode protein products essential for bacterial viability and are therefore subject to selective pressure. These DNA sequences are then compared. Based on the sequence differences or polymorphisms detected, each unique sequence is termed an allele and is identified by a unique sequence type (ST) number. ST numbers associated with these loci are then used to compare isolates and infer genetic relatedness. Bacterial isolates can be identified by a string of ST numbers and for two individual bacterial isolates where the same ST number strings occur, these isolates are confirmed as being indistinguishable by this method.

MLST protocols have been described for a variety of important veterinary pathogens. The method is easily standardized and detailed protocols have been described (www.mlst.net).

Recently, attempts have been made to develop MLST schemes that are based on virulence genes. Some multi-virulence locus sequence typing schemes are now available and can be applied to selected pathogens of veterinary importance.

Whole bacterial genome sequencing

Information obtained from genome sequencing projects can aid our understanding of infectious disease and microbial evolution. The first technical approach to determine the DNA sequence for a bacterium necessitated the construction of an extensive library of randomly generated DNA fragments. These short DNA fragments were then subjected to a high-throughput sequencing pipeline. Powerful bioinformatic computing tools were used to search this collection to identify overlapping sequences. These were then spliced together, until the complete genome of the bacterium was assembled.

Advances in sequencing technologies and computational analysis over the past few years have led to the development of faster analytical approaches, so-called next-generation sequencing technologies. These new methods significantly increase the volume of sequence data that can be evaluated. In addition, the methylation profile of a bacterial genome can be determined using single-molecule real-time DNA sequencing.

Surveillance strategies are now assessing metagenomics as a culture-independent method for identification of individual members of microbial populations, irrespective of source. This strategy aims to determine the sequence of all nucleic acids recovered from a sample of veterinary interest and it has the potential to revolutionize the detection of known pathogens and other microorganisms sharing the same ecological niche. Metagenomics offers the prospect of directly predicting antibiotic-resistant phenotypes along with the identification of virulence and other genes without the requirement for culturing bacteria.