Current and emerging technologies for genotyping Campylobacter

Current and emerging technologies for genotyping Campylobacter
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SUMMARY
Species of the Campylobacter have been the most infective bacteria and pathogens that have been reported in both developing and the already developed countries around the globe. Genotyping of these bacteria help in identification, protection and treatment of the effects of these bacterial infections. In genotyping of the Campylobacter species, different methods and techniques have used which include fla typing, ribotyping, Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), pulse field gel electrophoresis, random amplified polymorphic DNA and the Multilocus Sequence Typing (MLST).
In their effectiveness, these techniques offer different applications in terms of the complexity, size of the sample and the cost of acquiring and maintaining the equipments for genotyping. For instance, in dealing with short period outbreaks such as in chickens, fla typing is a faster and effective method used. Consequently, amplified fragment length polymorphism analysis and random amplified Polymorphic DNA are reliable for global genotyping and epidemiological researchers.
LITERATURE REVIEW
1 Introduction
Campylobacter species are among the most prevalent bacterial enteric pathogens in both developed and developing nations. This review will only focus on the genotypic methods. Genotyping methods have been to identify the strains causing outbreaks due to the bacteria Campylobacter. In their applications, the methods of genotyping the bacteria have been used to identify the causes of infections and therefore trace the transmissions in animals and in human beings.
The methods in this review include fla typing, ribotyping, Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), pulse field gel electrophoresis, random amplified polymorphic DNA and the Multilocus Sequence Typing (MLST). Their techniques in genotyping is deeply described, identifying their respective strengths and weaknesses in genotyping and what technological advances have been achieved and are being undertaken to improve their effectiveness. Moreover, global laboratory interactions amongst researchers in exchanging the data and having reference laboratories has and will help in having a formal standardized and normalized database that will be electronically available for portability.
1 Campylobacter
The Campylobacter is a genus that encompasses a family of bacteria that basically attacks and infects the gastrointestinal tracts of its host. Some of the pathogenic species of the bacteria include the Campylobacter jejuni and the Campylobacter coli. These two are pathogenic to both human beings and also in animals. The eventual outcome of their infections is common acute human bacterial enteritis (Wassenaar et al, 2000). The main cause of infections to human beings has been attributed to the eating and also being in contact with contaminated meat from poultry. Research has shown that the gastrointestinal tracts are infested by the Campylobacter bacteria.. Moreover, animals such as sheep, goats, pigs as well as cattle may also be infected by the bacteria Campylobacter species.
The infection of Campylobacter jejuni in humans is greatly prevalent but the knowledge of the pathogenicity of the bacterial strains and its infections is still widely limited. These results in the difficulty in identifying which strains of Campylobacter jejuni are specific to certain hosts or whether they may be liable to some infections on human beings.
2 Genotyping of Campylobacter
In trying to control the effects and spread in human beings, it's very essential to understand the epidemiology of Campylobacter. The main assumption made about the disease is that it is a primarily food borne disease since the evidential cases previously have reported Campylobacteriosis to contaminated meat from poultry. However, since Campylobacter is ubiquitous in the surrounding, some cases have been reported as sporadic and their outbreaks are scarce. As a result, tracing the source of the infection may thus deem a difficult task to handle (Wassenar et al, 2000).
The difficulty in identifying the sources and causes of Campylobacteriosis has been attributed to the deficiency of reliable methods for typing and identifying the responsible Campylobacter strains that result infections in humans. With the availability of these techniques and methods, the epidemiology of the Campylobacter species in food producing animals in the farm environments can be well studied.
1 Classification of Genotyping Methods
1 Comparative Methods
The comparative typing methods have mostly been used in investing the outbreak of bacteria in humans by specifying on the short period prevention in a closed system such as in school or in a restaurant. This technique focuses on typing an isolated small number collected over a short time. Consequently, these comparative methods are used identifying the related and unrelated strains (Struelens et al., 1998).
2 Library Typing Methods
In surveillance of the bacterial epidemiology, library typing methods are most suitable. The collection and characterization of large number of organisms over a long period of time utilize strain markers with a standardized nomenclature and with high reproducibility over time. The distinguishing factor in the library typing methods should be balanced with the evolutionary stability of the organisms so as to permit the dispersion of clones to be identified over time.
Another approach includes the genomic sequencing of limited number of isolates of the same species providing a wealth of data on the micro-organism biology. This approach is only suitable when it comes to identifying the differences in the contents of a gene, the effects and other biological characteristics. This type of approach enables the collection of novel probes and sequences of DNA that are related to the phenotypic traits and to identify the processes of exchange of genes by using gene sharing and other unrelated class of microbes.
For epidemiological purposes banding profiles representing 10% (or less) of the genome are normally recommended. Some of the methods for genotyping Campylobacter jejuni are described and discussed in the following section (Manfreda & De Cesare).
2 Ribotyping
Various forms of loci of rRNA gene (coding for 5S, 16S and 23S rRNA) are positioned at numerous locations in the Campylobacter chromosome. Due to the strong preservation of regions present at the rRNA genes and also in the availability of noncoded flanking locations, these genes are applicable for subtyping purposes.
The ribotyping technique is therefore based on ribosomal genes and the subsequent agarose gel electrophoresis of the digested genomic DNA which is then enhanced by the southern blot hybridization with a probe only applicable for rRNA genes. Despite Campylobacter species having only three copies of ribosomal gene, they have a limited ability of distinguishing these copies. For instance, ribotyping can not be used to identify Campylobacter fetus subspecies from Campylobacter fetus subspecies venerealis and to separate the strains amongst the subspecies (Denes et al, 1997). Therefore, it implies that ribotyping technique prove to be the most appropriate method to identify the species of Campylobacter that are deemed to be difficult to analyze phenotypically.
In most applications, a probe derived by Polymerase Chain Reaction (PCR) from Campylobacter DNA and specifically utilized the 16S rRNA. However, the limit in distinguishing ribotyping and other more elaborate nature of this method have made it to relatively unreliable for method for routine genotying. The first approach has been the rehybridization of the southern blots using probes that are positioned along the regions of the chromosome to increase the power of distinguishing. As a result, this method has been successfully utilized with an inserting the clone of the polymorphic lambda (Jackson et al, 1997). In this method, the use of a single probe resulted in high discriminatory power that was gathered by the probe of a ribosome since the previous probe contained a wider insertion area. However, the characteristics and form the lambda clone insert was unknown and therefore the sequence of the gene of Campylobacter jejuni strain that was available should have enabled a faster identification of the sequence of polymorphic that was observed.
A number of other probes used in addition with the ribosomal genes are pieces gathered from genes that form ATPase, ribosomal protein S12 and the elongation factor EF-Tu (Weijtens et al, 1997). The second improvisation involves riboprinting which is a technique that overcome the cumbersomeness and improves the reproducibility of this method. Moreover, riboprinting has been utilized in subtyping Campylobacter jejuni and Campylobacter coli strains that were collected from a variety of sources.
However, it is expensive to purchase the equipment for riboprinting and other consumables and is severely limited in its use even though the automated devices have been utilized for multiple bacteria.
3 Flagellin Typing (Fla Typing)
In this technique, the locus of the flagellin gene of the Campylobacter jejuni is comprised of two different genes which are flaA and flaB and are organized in tandem with a separation gap of approximately 170 nucleotides. These genes usually share a 92% homology and arranged in the Campylobacter genome. Moreover, they also exhibit approximately 95 % sequence variation between their isolates which provide them with a basis of fla typing schemes. Fla typing however involves PCR amplification of the entire flaA or flaB gene which is accompanied by the digestion by restriction enzymes that is present (Salama, 1992). Subsequently, polymerase chain reaction (PCR) amplifications are then directed to restriction enzyme digestion that results in PCR restriction fragment length polymorphism profiles that follows gel electrophoresis.
Due to the existence of a strong conservation in the strain connecting the flaA and flaB, there is a supplementation of a specific flaB primer that differentiates 10% of the total isolates (Mohran et al, 1996). There is the modernization of a new nested primer which simplifies the method regardless of its simplification and sensitivity. For instance, the recently created and adopted primers do overlap as shown in figure 1. In this overlap, only the forward primers replicate the origin of the flaA gene representing a small variation.
Consequently, the opposite primer (reverse) is often inconsistent in its applications. As a result of these variations in directions, the primer therefore affects the PCR/RFLP profiles that have been developed by the method.
Despite this, fla typing has been identified as reliable, suitable, and relatively simpler subtyping method, the variations in the primers and the processes do not allow the outcomes obtained in different reference laboratories to be directly compared and analyzed. However, since fla typing profiles and codes can be easily integrated into electronic databases, a wide range of isolates are analyzed in comparison to other global studies and databases for feasibility reasons (Penner & Hennessy, 1980).
Figure SEQ Figure \* ARABIC 1 shows a PCR analysis of the flagellin genes (fla typing) of Campylobacter jejuni. Figure (A) is a schematic illustration of the PCR primers applied in genotyping flaA. The numbers of the primers (arrows) corresponding to the reference numbers (Ref:) in panel C. the lengths of the amplified fragments are indicated. (B) Alignment of the first and last 37 nucleotides of all available flaA sequences (Meinersmann, 2002). (C) Primers for flaA amplification compared with a recommended consensus sequence derived from all previously published flaA sequences.
4 Multilocus Sequence Typing Scheme (MLST)
This technique was devised in the year 1998 as a novel approach to bacterial genotyping. The model utilized in its inception was a Neisseria meningitides. In its application, MLST involves PCR amplification and subsequent nucleotide sequencing of the internal pieces of the conserved housekeeping genes. In the sequencing process, seven of the housekeeping loci which are adversely distributed throughout the genome are sequenced by applying the MLST scheme which directs...