DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from caecal samples of chickens in Grenada

Authors


Omar A. Oyarzabal, Department of Poultry Science, Auburn University, 260 Lem Morrison Dr., AL 36849, USA. E-mail: oyarzoa@auburn.edu

Abstract

Aims:  To speciate Campylobacter strains from the caeca of chickens in Grenada using PCR and to evaluate DNA-based typing methods for the characterization of these isolates.

Methods and Results:  Isolates were speciated with two multiplex PCR assays and were typed with flaA-RFLP, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results confirmed that Campylobacter coli strains were more predominant than Campylobacter jejuni strains. From 56 isolates, 18 were misidentified using biochemical tests. PFGE typing gave the highest discriminatory power among the methods used (Simpson’s index of diversity, D = 0·9061). However, the combination of flaA-RFLP, PFGE and MLST results gave the highest discrimination for subtyping of these isolates (= 0·9857). A band position tolerance of 4% in BioNumerics was the most appropriate for the analysis of this database. MLST profiles were generally concordant with PFGE and/or flaA-RFLP types. Several isolates exhibited new MLST sequence types (STs), and 43 of the 49 Camp. coli strains belonged to the ST-828 clonal complex.

Conclusions: Campylobacter coli was the most prevalent species isolated from broilers and layers in Grenada, and a combination of restriction and sequence methods was most appropriate for the typing of Camp. coli isolates. Campylobacter coli STs clustered with described poultry-associated Camp. coli STs by phylogenetic analysis.

Significance and Impact of the Study:  Further studies to understand the predominance of Camp. coli within Campylobacter spp. from chickens in Grenada may help elucidate the epidemiology of these pathogens in chickens.

Introduction

The identification of Campylobacter jejuni and Campylobacter coli by biochemical testing is hindered by the inertness of these species and by the fact that they differ only by their ability to hydrolyse hippurate (Razi et al. 1981; Patton et al. 1991; Debruyne et al. 2008a,b). The limitations of biochemical tests have prompted the use of complementary molecular techniques to accurately differentiate between these two Campylobacter species. These techniques rely on amplification and/or sequencing of specific genes or gene segments.

In addition to the species identification, molecular techniques are used for determination of genetic relatedness of the Campylobacter isolates. The most common techniques used for subtyping of Campylobacter strains include amplification and restriction techniques, such as restriction fragment length polymorphism of the flaA gene (flaA-RFLP), restriction and migration techniques, such as pulsed-field gel electrophoresis (PFGE), and sequencing techniques, such as multilocus sequence typing (MLST) (On et al. 2008).

Grenada is an island nation in the south-eastern Caribbean Sea. In 2003, Grenada produced approximately 14% of the 7·2 million kg of chicken meat consumed on the island (David 2004) and imported the remaining 86%. An average of 25·4 kg per capita of chicken meat is consumed annually in Grenada (Anon. 2008). Information concerning human campylobacteriosis in Grenada is not readily available. Statistics have revealed that gastroenteritis and diarrhoea are among the leading causes of infant mortality between 1992 and 1996. In these years, all causes of gastroenteritis increased by 60% in children <5 years of age and by 73% in children over 5 years (Anon. 1998). As a result of the lack of information regarding the prevalence of Campylobacter in poultry meat in Grenada, we decided to analyse Campylobacter strains collected from broilers and layer chickens in Grenada.

The objectives of this study were to verify the identity of Campylobacter isolates using multiplex polymerase chain reaction assays (mPCR) and to determine the genetic relatedness of the isolates with flaA-RFLP, PFGE and MLST. Previously, these isolates were identified to the species level, using culture media and biochemical tests (Hariharan et al. 2009). The discrepancies between the results from biochemical tests and the results from mPCR assays, the degree of discrimination of flaA-RFLP, PFGE and MLST and the combination of these techniques are discussed.

Materials and methods

Collection and biochemical identification of isolates

Fifty-six Campylobacter isolates collected during processing from the caeca of broiler and layers were submitted to the Department of Poultry Science, Auburn University for DNA identification and characterization. These samples were collected from five farms and included 43 strains isolated from broilers and 13 strains isolated from layers. These samples were collected at each farm during processing (processing is performed manually in Grenada) on eight different days during the period of August 2006 to January 2007. The caeca were placed in sterile plastic bags and transported to the laboratory on ice for processing the same day.

The identification of these isolates using biochemical tests have been described elsewhere (Hariharan et al. 2009). Briefly, identification of the Campylobacter strains was performed as follows: freshly grown cultures were subjected to catalase, oxidase (BBL, Becton, Dickinson and Co., Sparks, MD, USA), hippurate (Remel, Lenexa, KS, USA) and latex agglutination tests (JCL, Panbio, Columbia, MD, USA). All isolates were also tested for their susceptibility to nalidixic acid (30 μg disc) and cephalothin (30 μg disc) on Mueller–Hinton agar supplemented with 5% sheep blood. All isolates were positive with the latex agglutination test. Based on biochemical tests, hippurate-positive isolates were identified as Camp. jejuni, while nalidixic acid-susceptible, hippurate-negative isolates were identified as Camp. coli, and nalidixic acid-resistant, hippurate-negative isolates were identified as Campylobacter lari (Hariharan et al. 2009).

Isolates were stored at −85°C in 2% sterile, skim milk in cryovials. From these stock cultures, strains were grown on blood agar plates (24 h at 42°C under microaerobic conditions) and the growth was used to inoculate tubes containing a mix of Cary–Blair and Brucella broth (1 : 3) supplemented with 5% lysed horse blood (20 ml final volume). These tubes were shipped under refrigeration (c. 8°C) to the Microbiology Laboratory at Auburn University, USA, for further studies. At Auburn University, isolates were recovered by plating the samples on modified Campy-Cefex (mCC, Oyarzabal et al. 2005). Samples were also enriched in Bolton broth (Oxoid, New York, NY, USA) for 24 h at 42°C under microaerobic conditions (10% CO2, 5% O2, and 85% N2; Airgas, Radnor, PA, USA) provided with an evacuation-replacement system (MACSmics Jar Gassing System; Microbiology International, Frederick, MD) and anaerobic jars. Enriched samples were swabbed (c. 0·1 ml) onto mCC plates. All plates were then incubated at 42°C under microaerobic conditions for 48 h and screened for typical Campylobacter colonies. Colonies were presumed positive if they showed typical morphology and motility under phase contrast microscopy (Optiphot-2; Nikon, Tokyo, Japan). Presumptive isolates were identified using API Campy tests (bioMerieux, Hazelwood, MO, USA), and DNA was extracted using PrepMan™ Ultra (Applied Biosystems, Foster City, CA, USA). Isolates were then stored at −80°C in tryptic soy broth (Difco, Detroit, MI, USA) supplemented with 30% glycerol (v/v) and 5% lysed horse blood for further identification and characterization.

PCR identification of Campylobacter isolates

DNA of Campylobacter isolates was tested using two multiplex PCR assays that were performed in 25 μl aliquots, as described elsewhere (Oyarzabal et al. 2005, 2007). These mPCRs selectively detect Camp. coli based on amplifications of the aspA (Linton et al. 1997) and ceuE (Gonzalez et al. 1997) genes, and Camp. jejuni based on the amplification of the hipO gene (Persson and Olsen 2005) and an undefined gene (Winters and Slavik 1995). The isolates biochemically classified as Camp. lari were tested with a PCR assay that uses primers that attach to hypervariable regions of the 16S rDNA and that has been shown to unambiguously identify Camp. lari (Oyarzabal et al. 1997). A PTC-100 Programmable Thermal Controller (Bio-Rad Laboratories, Hercules, CA, USA) was used for amplification. Amplicons were detected in 1·5% UltraPure™ Agarose-1000 (Invitrogen Corporation, Carlsbad, CA, USA). DNA identification of PCR bands and restricted DNA fragments of flaA-RFLP and PFGE were stained with ethidium bromide, visualized using a UV transilluminator (Gel-Doc System; Syngene, Frederick, MD, USA), and a digital picture was recorded using GeneSnap (Syngene).

flaA-RFLP characterization

Extracted DNA of Campylobacter isolates was tested for flaA-RFLP analysis, using 25 μl PCR reactions as described elsewhere (Nachamkin et al. 1993; Harrington et al. 2003). A PTC-100 Programmable Thermal Controller was used for amplification and the restriction enzyme was DdeI (New England Biolabs, Ipswich, MA, USA). DNA was restricted at 37°C for 2 h. After restriction, DNA bands were detected by electrophoresis in 2·5% SeaKem® Gold Agarose (Lonza, Basel, Switzerland).

PFGE characterization

We used a standard PFGE protocol (Ribot et al. 2001) with a few modifications described elsewhere (Oyarzabal et al. 2008). When collecting the cells to make the plugs, an OD600 nm of 1·0 was used instead of 0·57–0·82. Salmonella choleraesuis ssp. choleraesuis serotype Braenderup H9812 (ATCC BAA-664) restricted with XbaI was used as the DNA size marker. Campylobacter DNA was digested with SmaI and XmaI. The strains that did not show restriction with these enzymes were also tested with XbaI and BamHI. Restricted DNA fragments were separated with a contour-clamped homogeneous electric field (CHEF Mapper; Bio-Rad Laboratories) in 1% SeaKem® Gold Agarose gels that were stained subsequently with ethidium bromide. Visualization was carried out with a UV transilluminator (Gel-Doc System), and a digital picture was recorded using GeneSnap.

MLST characterization

MLST of all strains was performed using the Camp. coli primer sets described elsewhere (Miller et al. 2005). Each MLST amplification mixture contained: 50 ng genomic DNA, 1× MasterAmp PCR buffer (Epicentre, Madison, WI, USA), 1× MasterAmp PCR enhancer (Epicentre), 2·5 mmol l−1 MgCl2, 250 μmol l−1 (each) dNTPs, 50 pmol each primer and 1 U Taq polymerase (New England Biolabs, Beverly, MA, USA). MLST amplifications were performed on a Tetrad thermocycler (Bio-Rad Laboratories) with the following settings: 94°C, 30 s; 53°C, 30 s; 72°C, 2 min (30 cycles). Amplicons were purified on a BioRobot 8000 workstation (Qiagen, Valencia, CA, USA). Cycle sequencing reactions were performed on a Tetrad thermocycler, using the ABI PRISM BigDye terminator cycle sequencing kit (version 3.1; Applied Biosystems) and standard protocols. Cycle sequencing extension products were purified using BigDye XTerminator (Applied Biosystems). DNA sequencing was performed on an ABI PRISM 3730 DNA Analyzer (Applied Biosystems), using POP-7 polymer and ABI PRISM Genetic Analyzer Data Collection and ABI PRISM Genetic Analyzer Sequencing Analysis software. Alleles and sequence types (STs) were identified using the perl program MLSTparser (Miller et al. 2005). Novel alleles and STs were submitted to the PubMLST Camp. jejuni/Camp. coli database (http://pubmlst.org/campylobacter/).

Analysis of PFGE and flaA-RFLP results

TIF images of the restriction profiles for flaA-RFLP and PFGE were incorporated into BioNumerics ver. 4.50 (Applied Maths, Austin, TX, USA) for analysis. Pair-comparisons and cluster analyses were made using the Dice correlation coefficient and the unweighted pair group mathematical average (UPGMA) clustering algorithm. The optimization and position tolerance for band analysis were set at 4%, and a cut-off of 90% was used for the determination of the different restriction patterns for flaA-RFLP and PFGE. The Simpson’s index of diversity (Hunter and Gaston 1988) was used to determine the discriminating power of each restriction technique.

Results

Farm information

The age at slaughter was different, with broilers being processed at 6 weeks while layers were processed at 72 weeks of age. The number of Camp. coli isolates was higher than the number of Camp. jejuni isolates in samples from all farms, and Camp. jejuni represented no more than 33% of the isolates collected from samples from a given farm (Table 1, farm D).

Table 1.   Number and ID of the isolates collected form five farms in Granada
FarmSpeciesIDNo. isolates
  1. *Two isolates did not grow (not included in this count).

  2. †One isolate did not grow (not included in this count).

ABroilersCampylobacter coli18*
Campylobacter jejuni 2
LayersCamp. coli 7
Camp. jejuni 0
BBroilersCamp. coli 7
Camp. jejuni 0
DBroilersCamp. coli 4
Camp. jejuni 1
LayersCamp. coli 0
Camp. jejuni 1
EBroilersCamp. coli 9†
Camp. jejuni 2
KLayersCamp. coli 4†
Camp. jejuni 1

Identification of Campylobacter strains

From 60 cultures shipped to Auburn University, four were not recovered after enrichment. Therefore, 56 isolates were tested at Auburn University. The attempts to speciate these 56 isolates using the API test resulted in several ambiguous results. However, using two mPCR assays (Oyarzabal et al. 2005, 2007), 49 isolates were identified as Camp. coli, and seven were identified as Camp. jejuni. Isolates identified as Camp. lari (n = 3) by biochemical tests (Hariharan et al. 2009) were identified as Camp. coli by the mPCR assays and failed to generate amplicons in a PCR assay that uses primers specific for Camp. lari (Oyarzabal et al. 1997). In addition, 13 isolates identified as Camp. jejuni and two identified as Camp. coli by biochemical tests were identified as Camp. coli and Camp. jejuni respectively, with the mPCR assays.

Results from the mPCR assay showed that Camp. coli was the most prevalent species and that Camp. jejuni accounted for no more than 15% of the isolates. The higher prevalence of Camp. coli was strikingly similar in isolates collected from layers or from commercial broiler chickens (Table 2).

Table 2.   Origin of the isolates and distribution by species
OriginSpeciesNumberPercentage
BroilersCampylobacter jejuni512
Campylobacter coli3888
LayersCamp. jejuni215
Camp. coli1185
TotalCamp. jejuni712·5
Camp. coli4987·5

Two of these isolates (G185 and G187) gave mixed results (both Camp. jejuni and Camp. coli bands) during PCR analysis. These strains were re-isolated using a 0·65-μm filter (Millipore Corp., Billerica, MA, USA), and one isolated colony from each sample was analysed again by mPCR, flaA-RFLP and PFGE. G185 was identified as Camp. coli and gave a restriction profile with PFGE, while G187 was identified as Camp. jejuni but did not restrict with PFGE using SmaI, XmaI, XbaI or BamHI. In addition, isolate G194 was a mix of two Camp. coli strains. This mixed isolate was identified by ambiguous MLST results during sequencing and was confirmed by an unusually large number of PFGE bands. Again, filtration helped us isolate a pure Camp. coli strain from this mix.

flaA-RFLP and PFGE results

One isolate did not yield any flaA-RFLP profile using the described protocol. The remaining 55 isolates (Camp. jejuni and Camp. coli) gave 14 flaA-RFLP types. Several strains did not show any restriction by PFGE when plugs were made with an OD600 nm of 0·57–0·82 (Ribot et al. 2001), but these strains did exhibit restriction when plugs for PFGE were re-made using an OD600 nm of 1·0. At the end, one Camp. jejuni isolate and five Camp. coli isolates did not show any restriction by PFGE using SmaI or XmaI. The rest of the isolates gave 19 PFGE types. Five types accounted for 64% of the strains (34 strains), while the other 14 accounted for 21 strains (Fig. 1). Two Camp. coli strains (G102 and G151) that did not restrict with SmaI or XmaI showed a restriction profile with BamHI.

Figure 1.

 Pulsed-field gel electrophoresis (PFGE) restriction profiles of Campylobacter coli and Campylobacter jejuni isolates from Grenada. Pair comparison and cluster analysis performed with the Dice correlation coefficient and the unweighted pair group mathematical average clustering algorithm of BioNumerics ver. 4.50. The optimization and position tolerance for band analysis were set at 4%, and a cut-off of 90% was used for the determination of PFGE types. The right columns show the PFGE (letters), flaA-RFLP (roman numerals) and multilocus sequence typing (numbers) types for each isolate.

MLST results

The allelic profiles from the MLST analysis are presented in Table 3. Several isolates exhibited new STs, which were deposited in the Camp.  jejuni/Camp. coli PubMLST database. Additionally, the new allele tkt365 was identified in all Camp. coli ST-3841 isolates. Among the 49 Camp. coli isolates typed in this study, remarkably few alleles were identified, and only eight STs were identified: for two loci, atpA (uncA) and glyA, only one allele was present: atpA17 and glyA82, respectively; the tkt locus had the largest number of alleles with four identified. Although many of the Camp. coli alleles identified here are considered ‘common’ (i.e. nonhost-associated), chicken- or poultry-associated alleles were also identified within the Camp. coli profiles, consistent with their isolation from chickens. These alleles include gltA65, pgm113 and tkt47. No alleles associated previously with cattle or swine were identified in this study.

Table 3.   Allelic profiles and clonal complexes from 56 Campylobacter coli and Campylobacter jejuni strains isolated from chickens in Grenada
Allelic profilesClonal complexSTNo. of strainsCountrySourceIsolated from
  1. ST, sequence types.

  2. *Camp. jejuni STs in bold.

  3. †No clonal complex assigned yet.

KnownST-828163016CanadaHuman stoolBroilers and layers
8945USATurkeyBroilers
8251UK, SpainHuman stool, environmental water, and pigBroilers
11737USAChickenBroilers and layers
ST-443*511Netherlands, UK, Canada, Curaçao, USAHuman stool, chickenLayers
ST-3543542UK, Australia, Canada, USA, BelgiumHuman stool, human blood culture, chicken, chicken offal or meat, calf, sheepBroilers
ST-3533533UK, Curaçao, USA and CanadaHuman stool, human blood culture, chicken, chicken offal or meatBroilers and layers
ST-2578241UK and AustraliaHuman stoolBroilers
15816DenmarkHuman stoolBroilers and layers
NewST-82838419GrenadaChickenBroilers
38402GrenadaChickenLayers
38393GrenadaChickenLayers

Forty-three of the 49 Camp. coli strains were members of the ST-828 clonal complex; the remaining strains (ST-1581) were not associated with any clonal complex. Within the ST-828 clonal complex, ST-1630 included the largest number of strains and also the most numbers of PFGE and flaA-RFLP profiles (Table 4).

Table 4.   Pulsed-field gel electrophoresis (PFGE) and flaA-RFLP types and number of strains per each multilocus sequence typing (MLST) profile found in this study
SpeciesMLSTNo. of typesNo. of strains
PFGEflaA-RFLP
  1. *One strain failed to restrict with flaA-RFLP.

  2. †NR, no restriction. Four strains failed to restrict with PFGE.

  3. ‡One strain failed to restrict with PFGE.

Campylobacter coli3841A, BIII, IX*9
3840M, SI, VIII2
3839E, K, LIII, XIV3
1630C, F, G, H, J, NR†I, V, VI, VII16
1581D, NR‡I, II, VIII6
1173A, B, EI, II7
894A, BIII, X5
825GIX1
Campylobacter jejuni824PXI1
354I, NR‡III, IX2
353N, R, QIV, XII3
51OXIII1

Discriminatory power of the three typing methods

Twelve MLST STs were identified, with a Simpson’s index of diversity of 0·8636. Most of the major PFGE and flaA-RFLP clusters were concordant generally with ST, and contained strains with the same or phylogenetically similar STs. The combination of flaA-RFLP, PFGE and MLST results gave the highest discrimination for these isolates, with a total of 42 different combined types for 56 strains. The combination of results gave the highest Simpson’s index of diversity for these strains (Table 5).

Table 5.   Simpson’s index of diversity for each typing method and a combination of all methods used in this study
Typing method Simpson’s indexNo. of types
  1. MLST, multilocus sequence typing; PFGE, pulsed-field gel electrophoresis.

flaA-RFLP0·874114
MLST0·863612
PFGE0·906119
Combination (all three methods)0·985742

Discussion

The biochemical differentiation of Camp. jejuni and Campcoli is based on one test, hippurate hydrolysis (Debruyne et al. 2008a). Although all Camp. jejuni carry the hippuricase gene, some Camp.  jejuni strains do not express the enzyme for this reaction and appear to be hippurate negative. Therefore, the use of molecular methods for species identification should not be overlooked when trying to differentiate Camp. jejuni from Camp. coli. In our studies, the agreement between the two mPCRs was 100%. One of these mPCR assays target the ceuE gene and is specific for Camp. coli (Gonzalez et al. 1997; Nayak et al. 2005; Oyarzabal et al. 2007; Debruyne et al. 2008a), while the other mPCR reacts only with the aspartokinase gene of Camp. coli (Linton et al. 1997; Oyarzabal et al. 2005; Debruyne et al. 2008a). It is apparent that Camp. lari has not been documented in live chickens or commercial broiler meat, because of the incorporation of molecular techniques for routine identification of Campylobacter isolates.

Interestingly, in our results, Camp. coli made up the majority of the Campylobacter strains from broilers and layers in these samples from Grenada. No information was available for specific flocks, and therefore we could not link any flock to any given breed. Most reports from the rest of the world show that Camp. jejuni is the most predominant Campylobacter species in live, commercial broilers, and in retail broiler meat (Genigeorgis et al. 1986; Annan-Prah and Janc 1988; Hofshagen 2007; Oyarzabal et al. 2007). However, two studies have reported a different ratio between Camp. coli and Camp. jejuni for some flocks.

El-Shibiny et al. (2005) reported a higher prevalence of Camp. coli in free-range and organic chickens over 5 weeks of age. These authors identified their isolates using PCR protocols developed originally by Linton et al. (1997). Coincidently, we also used the primers targeting the aspA gene of Camp. coli from Linton et al. (1997) in one of our multiplex PCR assays. El-Shibiny et al. (2005) concluded that their results were related to the extended rearing period of the free-range chickens (56 days) and organic chickens (73 days), and to the possible exposure to more environmental sources than commercially raised chickens. In another report, Petersen et al. (2001) found one breeder flock, out of two tested, with higher Camp. coli prevalence (36%), and suggested that adult hens are more susceptible to colonization by Camp. coli. In our study, we did not find any correlation of age and Camp. coli presence, and the prevalence of Camp. coli in broilers and layer was similar. Because layers were much older birds (72 weeks) than broilers (6 weeks), we think that the most probable explanation for these results is that layers acquire Camp. coli from environmental sources. Because several farms reared both broilers and layers, it is also logical to think that layers may serve as the source of contamination for broilers.

Several ST numbers from MLST analysis were found in broilers and layers, and some have been already reported from other sources (Table 3). A previous study had identified putative host-associated Camp. coli MLST alleles and STs (Miller et al. 2006). Additionally, a phylogenetic analysis, using concatenated allele sequences as described previously (Stoddard et al. 2007), indicated that the Camp. coli STs identified in this study clustered with other poultry-associated Camp. coli STs present within the PubMLST database. These findings suggest again that exposure to unique environmental sources may account for this large percentage of Camp. coli isolates in layers and in broilers. We will continue some studies to identify the source of contamination for layers.

Campylobacter jejuni isolates grouped with Camp. coli isolates by flaA-RFLP, and one Camp. jejuni strain grouped within the Camp. coli by PFGE. However, all Camp. coli and Camp. jejuni isolates were readily distinguishable following MLST, with strains from the two species forming distinct groups. Additionally, the MLST speciation matched 100% with the mPCR results. It appears that the argument for the continuous differentiation between Camp. jejuni and Camp. coli is becoming more challenging, especially now that a strong convergence between these two species has been described (Sheppard et al. 2008). Although lateral DNA transfer between the two species has been reported (Meinersmann et al. 2003; Miller et al. 2006), the extent of exchange and the frequency of transfer are not fully known because studies usually focus on a small section of the whole genome.

The PFGE analysis was performed using variable band position tolerance (1–4%) in BioNumerics. The use of a less stringent position tolerance (1–2%) resulted in a larger number of types, while a more stringent position tolerance (3–4%) yielded results that were more concordant with a general analysis by visual inspection of macrorestriction profiles using accepted guidelines (Tenover et al. 1995). The number of reports about the influence of band tolerance settings in BioNumerics, for the analysis of large databases of Camp. coli isolates, is limited. Carriço et al. (2005) evaluated the PFGE profiles of 1798 isolates of Streptococcus pneumoniae with two coefficients, Dice and Jaccard coefficients, and tested different position tolerances for band analysis. These authors found that a 2% position tolerance was most appropriate for their database. Perhaps for the analysis of Camp. coli isolates, a more stringent position tolerance may be suitable to account for the genetic variability found by PFGE analysis. However, a larger number of Camp. coli isolates should be tested to validate this assumption.

Some isolates with similar PFGE profiles had different flaA-RFLP restriction patterns, or vice versa. Therefore, a combination of fingerprinting methods was necessary to achieve a higher discrimination of isolates. The characterization of Campylobacter isolates with flaA-RFLP has several advantages, such as low cost, simplicity and a good level of standardization among laboratories (Djordjevic et al., 2007). Using a 90% cut-off for the analysis of the results, we obtained more restriction profiles with PFGE than with flaA-RFLP. These results agree with other reports suggesting that PFGE using SmaI is usually more discriminatory for Camp. jejuni and Camp. coli isolates than flaA-RFLP (Newell et al. 2000; Han et al. 2007). Yet a combination of PFGE, a restriction profile technique, with MLST, a sequencing technique, provided the most discriminatory combination.

To summarize, our results emphasize that Camp. coli was the predominant Campylobacter spp. from broilers and layers in these samples from Grenada and that there is a need to complement the identification of isolates by biochemical tests with molecular methods if speciation is necessary. The choice of technique for fingerprinting sometimes relates to the techniques already in use in some laboratories. However, a thorough investigation should include at least two techniques, a restriction based and a sequenced based, to complement each other and yield more discriminatory results.

Acknowledgements

This publication made use of the Campylobacter jejuni Multi Locus Sequence Typing website (http://pubmlst.org/campylobacter/) developed by Keith Jolley and Man-Suen Chan and sited at the University of Oxford (Jolley et al., 2004, BMC Bioinformatics5, 86). The development of this site has been funded by the Wellcome Trust.

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