Fluoroquinolone Resistance Detection in Campylobacter coli and Campylobacter jejuni by Luminex ® xMAP™ Technology

Abstract

The proportion of Campylobacter spp. isolates that are resistant to fluoroquinolones, the drugs of choice for campylobacteriosis, has been increasing worldwide. We developed an innovative method based on a Luminex xMAP™ DNA suspension array that allows the identification of Campylobacter species and, simultaneously, the detection of the most common point mutation in the gyrA gene (substitution from threonine 86 to isoleucine 86) that is responsible for fluoroquinolone resistance. Ninety-six Campylobacter coli and Campylobacter jejuni isolates collected from turkeys were first investigated by microdilution test to characterize the antimicrobial resistance patterns. The isolates, amplified for the quinolone resistance determining region of the gyrA gene, were then tested using Luminex suspension array. The reliability of the method was demonstrated by the total concordance between the results obtained using Luminex and those of the sequencing of gyrA polymerase chain reaction products. The genotypic characterization of fluoroquinolone resistance using Luminex was also consistent with the data on phenotypical resistance obtained by microdilution test. The results of this study strongly support the potential of Luminex xMAP technology as an efficient molecular method for the rapid and accurate identification of C. coli and C. jejuni isolates and the characterization of the major determinant of fluoroquinolone resistance.

Introduction

C ampylobacter spp. is one of the major causes of enteritis and the most commonly reported zoonoses in humans in Europe since 2004 (EFSA, 2010). More than 90% of the cases of campylobacteriosis are caused by Campylobacter jejuni and most other cases are attributable to Campylobacter coli (Ogden et al., 2009). Although antimicrobial treatment is rarely indicated because the disease is usually self-limiting, when needed the drugs of choice are fluoroquinolones and macrolides (Luangtongkum et al., 2009). Resistance to these antimicrobials for both C. jejuni and C. coli has become a major problem worldwide (Cokal et al., 2009; Gu et al., 2009). Particularly, the emergence of antimicrobial resistance due to the use of antimicrobial agents in food animals is a matter of great concern due to the zoonotic relevance of Campylobacter (Aarestrup and Wegenre, 1999; Evans et al., 2009). With specific regard to fluoroquinolones, several mechanisms responsible for resistance have been described, such as decreased outer membrane permeability and efflux pump (Beilei et al., 2005; Lin et al., 2005). The most common of these mechanisms involves mutations in the DNA gyrase gene (or “DNA topoisomerase II”) and in the DNA topoisomerase IV gene (Aarestrup and Engberg, 2001). These genes encode large enzymatic quaternary structures that consist of two pairs of subunits: GyrA and GyrB (DNA gyrase) and ParC and ParE (DNA topoisomerase IV) (Payot et al., 2006). Because there has been increasing evidence that topoisomerase IV is absent from Campylobacter spp. (Bachoual et al., 2001; Luo et al., 2003), fluoroquinolone resistance may be mainly due to mutations in the DNA gyrase gene (gyrA gene), which encodes the DNA gyrase subunit A (GyrA) (Wang et al., 1993; Griggs et al., 1996; Cooper et al., 2002; Best et al., 2003). Therefore, the absence of a secondary target for fluoroquinolones indicates that a single modification in the gyrA gene is sufficient for producing a fluoroquinolone-resistant phenotype (Wang et al., 1993; Bachoual et al., 2001; Cooper et al., 2002; Luo et al., 2003; Piddock et al., 2003; Payot et al., 2006).

C. coli and C. jejuni are closely related species, sharing 86.6% identity at the nucleotide sequence level, and genetic exchange between them has been reported (Fitch et al., 2005; Kinana et al., 2007). In particular, a point mutation (C to T) that produces an amino acid substitution from threonine 86 (Thr₈₆) to isoleucine (Ile₈₆) in the quinolone resistance determining region (QRDR) of GyrA has been identified as the main mechanism of fluoroquinolone resistance shared by these species (Wang et al., 1993; Piddock et al., 2003; Jesse et al., 2006). Although numerous attempts have been made to characterize this genetic determinant using techniques such as real-time polymerase chain reaction (PCR) (Wilson et al., 2000), mismatch amplification mutation assay PCR (Zirnstein et al., 1999, 2000), and PCR–restriction fragment length polymorphism (Alonso et al., 2004), these methods cannot be easily adapted to a multiplex format (Hakanen et al., 2002). Moreover, direct sequencing, which has long been regarded as the gold standard for the identification of genetic variations and the determination of the nucleotide arrangement in a DNA fragment, is not economically sustainable in routine use.

Suspension array, a high-throughput method for nucleic acid detection, represents a valid alternative to this traditional approaches. In particular, Luminex xMAP™ technology, a specialized flow cytometer, is being increasingly used to simultaneously investigate different molecular targets for genotyping, gene expression profiling, and genetic disease screening (Yang et al., 2001; Wallace et al., 2003; Borucki et al., 2005; Bovers et al., 2007; Call et al., 2007; Tracz et al., 2007; Huang et al., 2008; Leblanc et al., 2009). Luminex xMAP technology uses up to 100 sets of beads, which are distinguishable thanks to their unique spectral address, allowing detection of up to 100 different targets in a single sample well. In brief, specific PCR products labeled with biotin are mixed with beads that have been coupled to sequence-specific probes. The PCR products are subsequently marked with Streptavidin-R-phycoerythrin that binds to the biotin on the amplicons. Each probe bead-set hybridizes to its specific PCR product, when present. A red laser identifies the spectral address of the color-coded beads and a green laser registers whether or not the probe has captured a target (Dunbar et al., 2003). Luminex method is performed in a 96-well plate format, hybridization takes approximately 1 hour, and each well of the plate is assayed in approximately 0.47 seconds. Hence, multiple samples can be quickly analyzed at once in the same session (Baums et al., 2007). Its major advantages are that it is easy to use, allows for flexibility in array preparation, provides rapid, high-throughput detection of multiple targets, and produces numeric data that can be easily collected and used for comparison purposes (Page et al., 2006). Compared to traditional microarray, Luminex xMAP technology and other suspension arrays rely on instruments with a lower cost, faster assay hybridization kinetics, and more flexibility in array preparation and are simple to use (Dunbar, 2006).

The objective of the present study was to develop a rapid and reliable assay to identify C. coli and C. jejuni isolates and to simultaneously detect the most common point mutation in the gyrA gene that is responsible for fluoroquinolone resistance. In particular, the test consists of a Luminex suspension array assay based on four-bead pools with four specific oligonucleotide probes designed to efficiently identify C. coli and C. jejuni isolates and characterize the missense mutation occurring within the QRDR of the gyrA gene responsible for an amino acid substitution at position 86 (pT86I). This preliminary study is aimed at assessing the feasibility of the application of suspension array test to Campylobacter characterization, to address future research to a greater exploitation of the multiplexing characteristics of this assay.

Materials and Methods

Bacterial isolates

Ninety-six Campylobacter strains (52 C. coli and 44 C. jejuni) isolated from turkeys slaughtered in different abattoirs in northeastern Italy from October 2005 to November 2006 were analyzed. Bacteriological isolation and identification of thermotolerant Campylobacter strains were carried out according to the ISO 10272: 2006 (Anonymous, 2006). Speciation of Campylobacter isolates was performed using a multiplex PCR, as previously described by Denis et al. (1999). As reference strains, C. coli American Type Culture Collection (ATCC) 11353 and C. jejuni ATCC 33560 were used.

Antimicrobial susceptibility testing

Campylobacter isolates were first tested for susceptibility to quinolone (nalidixic acid) and fluoroquinolone (ciprofloxacin), using a commercial microdilution test (DKMVC2 Sensititre^® Susceptibility Plates for Campylobacter TREK Diagnostic Systems) according to manufacturer's recommendations. The results were read visually after 48 hours of incubation, and the minimum inhibitory concentration was defined as the lowest concentration of the antimicrobial that completely inhibited visible growth. The resistance breakpoint for ciprofloxacin was that suggested by the European Food Safety Authority (EFSA, 2007), whereas for nalidixic acid it was based on the recommendations of the Clinical and Laboratory Standards Institute (CLSI, 2008).

DNA extraction and PCR conditions

The bacterial strains were grown on mCCDA plates produced adding selective supplement (SR0155; Oxoid Ltd.) to the Blood-Free Selective Agar Base (CM0739; Oxoid Ltd.). Bacterial cells were then harvested by scraping each plate with a loop and were dissolved in 200 μL of distilled water. Genomic DNA was extracted by the conventional boiling method (Bachoual et al., 2001). The cell suspension was boiled at 100°C for 20 minutes and then centrifuged for 2 minutes at 14,000 g. The boiled cell debris was pelleted, and the supernatant was removed and used as DNA template in PCR.

A fragment of the QRDR in the gyrA gene of both C. jejuni and C. coli (codons 29–127) was amplified by PCR using the primers QRDR-FW and QRDR-RV-biotinylated, previously described by Niwa et al. (2003), using degenerated bases corresponding to polymorphisms occurring between C. jejuni and C. coli (Table 1). Standard PCR was performed with iProof™ High-Fidelity DNA polymerase (Bio-Rad Laboratories) following the manufacturer's instructions and using an ABI GeneAmp 9700 thermalcycler. The PCR conditions were as follows: initial denaturation at 98°C for 2 minutes, 35 cycles of denaturation at 98°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 1 minutes, with a final extension at 72°C for 5 minutes. Five microliters of template DNA (100–200 ng) was added to the PCR mixture (45 μL), which consisted of 1X iProof HF Buffer, 0.2 mM deoxynucloside triphosphates mix, 1 U High-Fidelity iProof DNA polymerase, and 0.4 pmol of each primer/μl. PCR products were observed using 2.0% agarose electrophoresis and ethidium bromide staining. The amplicons were seen as a band of 286 bp; no bands were detected in the negative controls.

Table 1.

Primers and Probes Used in This Study

Target	Primer/probe	Sequence (5′ to 3′)
Campylobacter spp.	QRDR-F^a	GAGYGTTATTATMGGTCGTGC^b
	QRDR-R^a	Bio-TTCAGTATAACGCATYGCAGC^b
C. coli wild type	cThr86wt^c	GCGATACTGCTGTTT
C. coli mutated	cThr86Ile^c	GCGATATTGCTGTTT
C. jejuni wild type	jThr86wt^c	GGAGATACAGCAGTTTAT
C. jejuni mutated	jThr86Ile^c	TGGAGATATAGCAGTTTA

Primers.

Sequence primer previously described by Niwa et al. (2003), with the exception that a single-nucleotide polymorphism (Y) occurring between published C. coli and C. jejuni sequences was considered.

Probes.

QRDR, quinolone resistance determining region.

The specificity of the amplified fragments was confirmed by direct sequencing of both sense and antisense strands of PCR products using an ABI 377 DNA sequencer (Applied Biosystems). DNA sequence editing, alignment, and analysis were performed with the BLASTN program (Altschul et al., 1997). Sample sequences were then compared to the previously published C. coli and C. jejuni sequences (GenBank accession numbers AF092101 and L04566 for the C. coli and C. jejuni gyrA genes, respectively) by alignment with Multalin Expression software (Corpet, 1988).

Thirty-six non-Campylobacter strains were used to perform the exclusivity test (Table 2).

Table 2.

Non-Campylobacter Strains (36) Used for the Exclusivity Test

Species	No. of isolates	Source
Salmonella enterica serotype Enteritidis	1
S. enterica serotype Typhimurium	1
S. enterica serotype Livingstone	1
S. enterica serotype Newport	1
S. enterica serotype Infantis	1
S. enterica serotype Havana	1
S. enterica serotype Anatum	1
S. enterica serotype 4,5:i:-	1
S. enterica serotype Bredeney	1
S. enterica serotype Blockley	1	IZSVe Laboratory repository
S. enterica serotype Heidelberg	1
S. enterica serotype Virchow	1
S. enterica serotype Arechavaleta	1
S. enterica serotype Javiana	1
S. enterica serotype Manchester	1
S. enterica serotype London	1
S. enterica serotype Kottbus	1
S. enterica serotype Abortusovis	1
Escherichia coli O157	1	IZSVe Laboratory repository
E. ATCC 25922	1	ATCC
E. coli ATCC 35218	1	ATCC
E. coli (clinical isolates)	6	IZSVe Laboratory repository
Yersinia enterocolitica	1	IZSVe Laboratory repository
Vibrio vulnificus ATCC 33815	1	ATCC
Vibrio alginolyticus ATCC 17749	1	ATCC
Vibrio proteolyticus ATCC 15338	1	ATCC
Vibrio mimicus ATCC 33654	1	ATCC
Vibrio fluvialis NCTC 11327	1	NCTC
Bacillus subtilis ATCC 6633	1	ATCC
Aeromonas sobria	1	IZSVe Laboratory repository
Shigella flexneri	1	IZSVe Laboratory repository

ATCC, American Type Culture Collection; NCTC, National Collection of Type Cultures; IZSVe, Istituto Zooprofilattico Sperimentale delle Venezie, Italy.

Coupling of oligonucleotide probes to beads

Four oligonucleotide probes (Table 1), previously described by Niwa et al. (2003), were used to detect the Thr86Ile mutation associated with resistance to fluoroquinolones in the gyrA gene of C. coli (cThr86wt, cThr86Ile) and C. jejuni (jThr86wt, jThr86Ile). The coupling was performed using a carbodiimide-based procedure (Luminex Corporation), following the manufacturer's instructions. The concentration of the coupled beads was quantified by hemacytometer counting. The efficiency of the coupling of new bead batches was verified using biotinylated oligonucleotide fragments (5–500 fmol) complementary to each probe. Efficient hybridization between the complementary fragments and the new coupled bead batches was tested with Luminex suspension array.

Multiplex DNA suspension microarray

The probe-bead stocks were diluted in 1.5 × tetramethyl ammonium chloride hybridization buffer so that each coupled bead was at a concentration of 150 beads/μL in the pool. For the assay, 33 μL of diluted bead pool (containing ∼5000 coupled beads of each set) was added to 5 μL of PCR product and 12 μL of TE buffer in duplicate in 96-well microtiter plates (Millipore). Each assay also included the analysis of a PCR-negative control that included all components except the amplicon target. The reaction mixture (50 μL total for each well) was incubated for 5 minutes at 94°C to denaturate targets and for 1 hour at 42°C to allow hybridization between coupled beads and PCR amplicons. The amplicons hybridized to coupled beads were centrifugated at 2250 g for 3 minutes and the supernatant was carefully removed. They were then resuspended in 75 μL of detection buffer containing reporter molecule (Streptavidin-R-phycoerythrin [Invitrogen] diluted to 2 ng/μL in 1 × tetramethyl ammonium chloride). The samples were incubated for an additional 5 minutes at hybridization temperature and then analyzed on the Luminex 100 analyzer (Bio-Plex™ 200 system instrument; Bio-Rad Laboratories). For each bead type in a given sample well, the raw data were expressed as the median fluorescent intensity (MFI), calculated from the signals of at least 100 beads. The reactions were analyzed with Bio-Plex Manager™ software (version 4.1.1). The positive/negative (P/N) ratio was determined by dividing the MFI of each isolate for a given bead set by the MFI obtained for the PCR-negative control. A minimum P/N ratio of greater than 3.0 was considered as the cut-off for defining positive reactions. The hybridizations of the biotin-labeled gyrA target DNA strands to the coupled beads and the Luminex analyses were performed in three independent experiments.

Results

Fluoroquinolone resistance

Ciprofloxacin resistance was shown for 25 (56.2%) of the 44 C. jejuni isolates and 43 (82.7%) of the 52 C. coli isolates. In all cases, the isolates that were resistant to ciprofloxacin were also resistant to nalidixic acid, demonstrating complete agreement between the two antimicrobials, which is consistent with their sharing the same mechanism of action.

DNA suspension array data output

Biotinylated PCR products for the QRDR were obtained from all 96 strains and the amplicons were then hybridized in the suspension array. The assay was first performed in uniplex (one coupled bead type); the four bead sets were then pooled in multiplex. Although the performance of the assay was comparable for the uniplex and multiplex formats, the MFIs in uniplex were generally higher than those in multiplex (data not shown). However, it is to point out that the results for the multiplex format were easily interpretable such as for the uniplex format.

The cThr86Ile and cThr86wt oligonucleotide probes successfully hybridized to all C. coli resistant and susceptible DNA targets, respectively. In particular, for resistant and susceptible strains, positive P/N ratios ranging from, respectively, 3.7 to 15.5 and from 3.5 to 6.2 were obtained for their specific probes. Similarly, all C. jejuni resistant and susceptible isolates hybridized with Jthr86ile and Jthr86wt probes, respectively. In particular, the DNA targets of the resistant strains generated positive P/N ratios ranging from 4.7 to 20.3 with the Jthr86ile probe, and the DNA targets of the susceptible strains produced P/N values ranging from 6.8 to 27.4 with the Jthr86wt probe. The hybridization-positive signals generated by the C. jejuni isolates that were susceptible to fluoroquinolones were generally higher than the signals generated by the resistant C. jejuni isolates and the C. coli isolates (both susceptible and resistant). The median P/N ratio for the negative signals produced by the four probe sets was 1.06. Thus, hybridization-positive signals were clearly distinguishable from nonspecific negative signals.

The suspension array was consistently successful in differentiating C. coli and C. jejuni isolates. Further, in resistant C. coli and C. jejuni isolates, the QRDR always showed the point mutation pT86I, whereas in susceptible isolates the target region was conserved. Representative P/N ratios averaged for two distinct isolates for each specific coupled bead are shown in Figure 1.

FIG. 1.

Representative positive/negative (P/N) ratios averaged for two distinct isolates for each specific coupled bead.

Concerning the exclusivity test, all 36 non-Campylobacter strains yielded negative results by both PCR assay for QRDR fragment and Luminex detection.

Sequence analysis

Comparative analyses between the sequences of the QRDR PCR products and the reference sequences (C. coli ATCC 11353, C. jejuni ATCC 33560) were performed to discriminate isolates of C. coli and C. jejuni and to identify the mutation occurring on QRDR of the gyrA gene (pT86I). Multiple sequence alignments showed that all strains, recognized by microdilution test as fluoroquinolone-resistant, presented the point mutation C to T at position 86. By contrast, no susceptible isolates showed the mutation at codon 86. Moreover, the identification of Campylobacter species and the discrimination of susceptible and resistant isolates based on sequence analysis were completely consistent with the data provided by Luminex suspension array (Table 3).

Table 3.

Characteristics of Campylobacter (96) Isolates as Shown by Microdilution Test, Luminex Suspension Array, and Sequence Analysis

		MIC (μg/mL) ^a		Luminex suspension array		Sequence analysis
Species	No. of isolates	Cip	Nal	C. coli/C. jejuni	Wild type/mutated	codon 86 ^b
C. coli	43	R	R	C. coli	Mutated	ATT
C. coli	9	S	S	C. coli	Wild type	ACT
C. jejuni	25	R	R	C. jejuni	Mutated	ATA
C. jejuni	19	S	S	C. jejuni	Wild type	ACA

The resistance (R) or sensitivity (S) against ciprofloxacin (Cip) and nalidixic acid (Nal) is indicated.

Observed mutations are shown in bold.

MIC, minimum inhibitory concentration.

Discussion

The method developed in this study appears effective for simultaneously identifying C. coli and C. jejuni and genotyping point mutation occurring on the codon that encodes the 86th amino acid of the GyrA protein (pT86I), using a multiplexed bead suspension array. Four oligonucleotide-specific probes, characterized by a single-base mismatch at the centre of their sequences, were tested in a single-well reaction with 96 C. coli and C. jejuni isolates. Each isolate was phenotypically characterized as “susceptible” or “resistant” to fluoroquinolone by microdilution test. On the basis of the probe-specific hybridization of gyrA gene amplicons, Campylobacter strains were speciated and discriminated unambiguously for the missense mutation at position 86, which is thought to be mainly responsible for fluoroquinolone resistance. The MFIs obtained in this study were generally lower than those reported in other works in which suspension bead array was used (Hakanen et al., 2002; Bovers et al., 2007; Ivanova et al., 2008; Eriksson et al., 2009; Leblanc et al., 2009). This is probably related to the choice of probes, which are characterized by few mismatches both among themselves and compared to the target sequences. Nonetheless, for all of the isolates the positive fluorescence signals were clearly distinguishable from the background and no ambiguous signals were found. The established threshold allowed to successfully recognize the Campylobacter species and to detect the point mutation resulting in amino acid replacement at codon 86 (pT86I) of the gyrA gene.

Comparative sequence analysis of the amplified fragments of the QRDR showed complete concordance with data obtained by multiplexed bead suspension array. Moreover, the multialignment sequence analysis confirmed that the missense mutation substituting Thr₈₆ for Ile₈₆ (pT86I) was identified in all isolates characterized as fluoroquinolone resistant by the microdilution test. Similarly, the strains that were phenotypically characterized as susceptible showed the wild-type sequence for the target region.

Although numerous mutations occurring at the gyrA gene and conferring fluoroquinolone resistance in Campylobacter spp. have been described (Jesse et al., 2006; Kinana et al., 2007), in this study a constant and close correlation between the phenotypic fluoroquinolone resistance and the presence of the T86I mutation in C. coli and C. jejuni was found. These results support previous evidence of the key role of the T86I amino acidic substitution in Campylobacter fluoroquinolone resistance (Jesse et al., 2006; Sonnevend et al., 2006). The epidemiological heterogeneity of the strains tested suggests that this evidence is unlikely due to their clonal origin.

It must be stressed that the method allows the detection of known mutations and is not suited to find unknown ones. Nevertheless, unlike other molecular approaches, such as PCR-based techniques, this method presents the main advantage of multiplexability, allowing a matrix of targets to be investigated simultaneously in the same sample. Further, the potential of multiplexing of Luminex xMAP technology could be further applied to develop this Campylobacter assay, studying also other molecular targets, such as different determinants of resistance to fluoroquinolone, and to macrolides as well as virulence and/or pathogenicity determinants. More generally, the quantity and accuracy of the data obtained with the assay can therefore be continually improved by adding probes to the array, presumably without any significant increase in the complexity of the analysis or the cost.

Footnotes

Acknowledgments

We thank the reviewers for their helpful contributions to the article. We are especially grateful to Douglas Call and his staff for the training provided and technical assistance, and to Mark Kanieff for his helpful comments and linguistic revision. This work was in part supported by grants from the Ministry of Health (project code IZSVe RC 03/06).

Disclosure Statement

No competing financial interests exist.

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