Phenotyping

Phenotyping ofCampylobacterincludes biotyping, serotyping and phage typing (Fitzger-

ald et al. 2001). Subtype or strain characterisation using phenotyping techniques becomes difficult and often ambiguous due to various reasons such as the lack of specific antisera when serotyping is employed; when there is unavailability of standard reagents; due to the presence of cross-reactivity between strains; and because of the emergence of high proportions of non-typeable strains (Jackson et al. 1996). Some of the widely used phe- notyping techniques are described below.

Biotyping

A biotyping scheme was first developed utilising 12 biochemical tests forCampylobac-

ter spp. that included growth at 28oC, hippurate analysis and resistotyping tests (using

antimicrobials or antibacterials) (Bolton et al. 1984). Generally the biotyping technique assesses the ability of the organism to utilise biochemical substrates and to grow in a dif- ficult environment, for example in the presence of an antibiotic (Klena 2001). Although these methods are advantageous in terms of their ease of use, time, and interpretation,

the inferences drawn from these tests are found to be too general for subtypingCampy-

lobacter(Struelens & Members of European Study Group on Epidemiological Markers

(ESGEM) of the European Society for Clinical Microbiology & Infectious Diseases (ES- CMID) 1996). The reproducibility and stability of these methods are not very good and have low discriminatory power and hence are often used with serotyping to make the scheme more useful (Sails et al. 2003b). However, antibiotic sensitivity testing (or an- tibacterial resistance testing) has outgrown other biotyping techniques in recent years and has been established as a ideal typing tool in its own right.

Antibiotic resistance typing is often referred to as resistotyping. This involves testing an organism to examine its sensitivity or resistance to selected antibiotics (Klena 2001). The organism that shows resistance to antibiotics are known as resistotypes determined using agar dilution (Bolton et al. 1984, Huysmans & Turnidge 1997) or disc diffusion methods (Lior 1984). This is a component of biotyping that measures the phenotypic trait expressed by the organism for which there may be numerous reasons such as a mutation in a gene that codes for antibiotic sensitivity or resistance. For example, mutations in the

Campylobacter gyrA gene have been shown to confer antibiotic resistance (Nachamkin

& Blaser 2000, chap. 2).

A cross-sectional study was conducted in Nebraska, United Sates of America to determine

the microbial profile and antibiotic susceptibility forCampylobacterspp. (Sanchez et al.

2002). This study evaluated the effect of immersion chilling and air chilling on micro- bial load on post-processed chicken carcases and found that immersion-chilled broilers

had a higher incidence of Campylobacter spp. resistance to nalidixic acid (NAL) and

other related fluoroquinolones, compared with isolates from air-chilled broilers. Similar studies on antibacterial and antimicrobial profiles have been conducted in various coun-

2.4 Typing techniques 25

tries such as The Netherlands (Oza et al. 2003), United Sates of America (Luangtongkum et al. 2007), Trinidad (Rodrigo et al. 2007), India and Iran (Baserisalehi et al. 2007) and Ethiopia (Dadi & Asrat 2009).

The most frequent pattern of antibiotic resistance, multi-drug resistance (MDR) inCampy-

lobacterspp. from imported chicken and human clinical cases was determined in a cross-

sectional study conducted in South Korea (Ku et al. 2011). Ku et al. (2011) found a

pattern of MDR resistance inCampylobacterspp. to four antimicrobials: ciprofloxacin,

nalidixic acid, ampicillin, and tetracycline. These findings indicate the extent of MDR

amongCampylobacterspp. in Korea; it is plausible that similar patterns might also exist

in other countries.

Serotyping

Serotyping ofCampylobacterwas initially based on the heat stable antigens (O) first de-

scribed by Penner & Hennessy in 1980. This was later adopted as the gold standard

for typing Campylobacterbeyond the species level (Penner & Hennessy 1980). Subse-

quently, there was another typing scheme developed by Lior et al. based on heat labile antigens (Lior et al. 1982). Of these two schemes, the Penner typing scheme is the most frequently used technique in laboratories worldwide and has undergone further develop-

ment with 66 different antisera being used for bothC. jejuni andC. colityping (McKay

et al. 2001). Although Penner serotyping was considered the gold standard, the exact na- ture of the serotyping antigen was not known at the time the technique was first developed (Moran & Penner 1999). Later it was discovered that the capsular polysaccharide (CPS)

was the contributory molecule for the serological reactions. GenerallyC. jejuniproduces

two different polysaccharide molecules; high molecular weight lipo-polisaccharide (LPS) and low molecular weight lipo-oligosaccharide (LOS) that contribute to the serological re- actions (Moran & Penner 1999). Karyshev and his co-scientists discovered that genes in the CPS region contain intragenic homopolymeric tracts, which are likely to render their expression phase variable enabling rapid antigen variation of the capsular polysaccharide (Frost et al. 1998, Karlyshev et al. 2000). Therefore the serological typing is thought to suffer from limitations such as lack of discrimination and cross reactivity (Frost et al. 1998, Karlyshev et al. 2000). Often Penner serotyping is used in conjunction with other methods due to its low discriminatory power and also due to the lack of antisera stan-

dardisation. In these circumstances new serotypes remain untyped (Frost et al. 1998, Wassenaar & Newell 2000).

Phage typing

Due to the low discriminatory power of serotyping, phagetyping was initially performed

to characteriseC. jejuniandC. coliusing 46 phage types (Grajewski et al. 1985, Khakhria

& Lior 1992). Phagetyping is often used as an adjunct to serotyping and about 76 defined phage-types have been defined to date (Nachamkin & Blaser 2000, chap. 2). Briefly, the technique ultilises a set of virulent phages on a bacterial host irrespective of any receptors for attachment. If the phages are capable of attaching and infecting the bacterial hosts, they lyse the bacterial cells producing a characteristic lytic pattern on the cultured petri dishes, referred to as ‘plaques (Grajewski et al. 1985). A phage type is defined as two or more epidemiologically unrelated isolates giving the same phage reaction pattern (Frost et al. 1999, Nachamkin & Blaser 2000). Like serotyping, the usefulness of phagetyping is also limited by the occurrence of non-typeable isolates and problems with cross reac- tivity (Sails et al. 2003b). This technique is labour intensive and expensive rendering it unsuitable for most clinical laboratories.

Multi-locus enzyme electrophoresis

Multilocus enzyme electrophoresis (MLEE) has been used as the standard typing tech- nique for eukaryotic population genetic studies (Ayala 1976) and was subsequently adapted to a variety of bacterial species to assess population diversity and the structure of bacterial

populations includingC. jejuni(Selander et al. 1986). This technique exploits the relative

electrophoretic mobilities of a large number of water soluble cellular enzymes (Selander et al. 1986). The rate of migration of a given protein in an electric field is dependent on amino acid sequences. The mobility variants of an enzyme is then directly equated with the alleles at the corresponding structural gene locus. Studies have shown that elec- trophoresis can detect large proportions of amino acid substitutions, however some silent substitutions may not be evident phenotypically and may not be discriminatory (Selander et al. 1986). Multiple enzymes encoded by housekeeping genes are analysed simulta- neously by MLEE (Maiden et al. 1998, Sails et al. 2003b). MLEE studies have been

2.4 Typing techniques 27

performed to determine the clonal framework of C. jejuni (Meinersmann et al. 2002).

This has provided insight into the nature of genome re-assortment and random exchange

of DNA segments that contribute to the genetic diversity ofC. jejuni. A number of clonal

groups have also been reported within theC. jejunipopulation (Meinersmann et al. 2002).

MLEE has also been utilised to study the congruence between other typing schemes used

forC. jejuni(such as multilocus sequence typing [MLST] and pulse field gel electrophore-

sis [PFGE]) (Sails et al. 2003b). However, MLEE does suffer from a number of limita- tions: (1) it examines the electrophoretic mobility of enzymes rather than indexing the molecular source of variation and hence may not be comparable between laboratories; (2) maintenance of live cultures may be time consuming and costly and mutations may occur if isolates are subcultured or stored for long periods of time; (3) this technique is time consuming, expensive and requires a high level of technical expertise (Sails et al. 2003b). All of these factors have rendered MLEE unsuitable for regular typing. MLEE has been superseded by a nucleotide-based technique, MLST which essentially mimics the MLEE’s multi loci principle.