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1 Department of Medical Microbiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates
2 Department of Medical Microbiology and Immunology, University of Pécs, Pécs, Hungary
3 Department of Medical Microbiology, Faculty of Medicine, University of Kuwait, Kuwait
4 Zoonoses and Emerging Infections Group, Clinical Virology, Clinical Department of Diagnostic Imaging, Infectious Diseases and Clinical Pathology, University of Veterinary Medicine, Vienna, Austria
Correspondence
Tibor Pál
Tibor.Pal{at}aok.pte.hu
Received 29 May 2006
Accepted 8 August 2006
Abbreviations: MAMA, mismatch amplification mutation assay; MT, molecular type; QRDR, quinolone resistance determinant region; UAE, United Arab Emirates.
The GenBank/EMBL/DDBJ accession numbers for the partial sequences of the gyrA genes representing new allelic variants are DQ449657, DQ449658, DQ449659, DQ449660, DQ449661, DQ449662, DQ449663 and DQ449664.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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While C. jejuni infections usually present as acute self-limiting gastrointestinal illnesses, in severe or prolonged cases antibiotic therapy is indicated, with macrolides as the primary drug of choice (Butzler, 2004). However, particularly if the causative agent is not identified, as is often the case in travellers' diarrhoea, fluoroquinolones are frequently used empirically (Guerrant et al., 2001; Yates, 2005). In such cases, resistance to ciprofloxacin can result in therapeutic failure (Guerrant et al., 2001; Sanders et al., 2002). Moreover, by mechanisms yet to be elucidated, fluoroquinolone resistance appears to coincide with increased fitness of the organism. In humans, fluoroquinolone-resistant strains are shed longer than sensitive isolates (Nelson et al., 2004; Engberg et al., 2004), and they have been reported to out-compete their sensitive counterparts, at least in certain genetic backgrounds, when colonizing poultry, i.e. the natural reservoir of the pathogen (Luo et al., 2005). All these findings underscore the necessity for close monitoring of the incidence and spread of fluoroquinolone-resistant clones of C. jejuni.
Countries on the Arabian Peninsula, some of them increasingly popular tourist destinations, have recently been scored as areas of high risk to acquire campylobacter infections (Ekdahl & Andersson, 2004). However, data on the incidence of fluoroquinolone resistance and on the type of strains present in the region are very limited (Sjogren et al., 1989; Albert et al., 2005; Jumaa & Neringer, 2005). The aim of the current study was to investigate the variety of C. jejuni strains isolated between 2002 and 2004 in a tertiary care hospital in Abu Dhabi Emirate, United Arab Emirates (UAE), and to determine the frequency and the genetic background of fluoroquinolone resistance among the local isolates.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Antibiotic susceptibility testing. The MICs of nalidixic acid, ciprofloxacin and erythromycin were determined by the agar dilution method using Mueller–Hinton agar (Oxoid) supplemented with 5 % (v/v) sheep blood and incubated under microaerophilic conditions at 36 °C for 48 h. The final antibiotic concentrations in the agar plates ranged from 0.25 to 512 mg l–1, according to the standards of the Clinical and Laboratory Standards Institute (CLSI; formerly known as the National Committee for Clinical Laboratory Standards, NCCLS) (NCCLS, 2003, 2005). Reference strain C. jejuni ATCC 33560 was included in each run as control.
Serotyping. Serotyping, based on the heat-stable antigen of the strains, was carried out by passive haemagglutination (Penner & Hennessy, 1980) using a set of commercial antisera (Denka Seiken) according to the manufacturer's instructions.
DNA purification. Genomic DNA was purified with a QIAamp DNA Mini kit (Qiagen) according to the manufacturer's instructions.
Mismatch amplification mutation assay (MAMA) PCR and sequencing of the QRDR of gyrase A. The MAMA PCR detection of the Thr-86 to Ile mutation in the gyrA gene of the quinolone-resistant isolates was carried out according to the method of Zirnstein et al. (1999). A 673-bp-long part of the gyrA gene, containing the QRDR, was amplified using primers GZgyrA5 and GZgyrA6 (Zirnstein et al., 1999), and directly sequenced by fluorescence-based direct sequencing employing the ABI Prism Dye Terminator cycle sequencing ready reaction kit (Perkin-Elmer) and an ABI Prism 310 genetic analyser automated sequencing system (Perkin-Elmer). For the analysis of the sequences, the program MEGA version 3.1 (Kumar et al., 2004) was used.
PCR-RFLP. The amplification of the flaA gene using consensus primers, and the digestion of the amplicon with DdeI were carried out as described by Wassenaar & Newell (2000). The digested products were run in a 2 % agarose gel at 1 V cm–1 for 120 min. Photographs of gels stained with ethidium bromide were analysed by the GelCompare II software (Applied Maths).
PFGE. C. jejuni genomic DNA was digested with SmaI following the formaldehyde inactivation of DNase, and the fragments were subsequently separated on a CHEF DRII apparatus (Bio-Rad) as described by the ‘Campynet’ PFGE Subtyping Subgroup (2000). The macrorestriction patterns of the isolates were compared according to the Dice similarity index (1-1 % tolerance interval) using the GelCompare II software. A PFGE group was arbitrarily defined as a strain if it exhibited macrorestriction band patterns with at least 95 % similarity.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Although fluoroquinolone resistance in campylobacter is rapidly emerging all over the world, high incidences comparable to the one found in our study have only been reported from a few locations, such as 88 and 75 % in Spain (Ruiz et al., 1998; Saenz et al., 2000), 96 % in Thailand (Sanders et al., 2002), 85.9 % in Hong Kong (Chu et al., 2004), and 77.1 % in India (Jain et al., 2005). Data from the Middle East are sparse. In 1998, Talhouk et al. (1998) reported a considerably lower level of ciprofloxacin resistance (39 %) in Lebanon among strains of human and animal origin. Recently, Albert et al. (2005) found 53 % ciprofloxacin resistance among human campylobacter isolates in Kuwait. It should be noted that the results of both the Kuwaiti study and our study were based on strains collected at single hospital laboratories. Further, more extensive, larger-scale studies should reveal whether or not the current studies reflect real differences in the incidences of fluoroquinolone resistance in this pathogen between the northern, and south-eastern parts of the Arabian Peninsula.
Earlier, Jumaa & Neringer (2005) had reported a 50 % fluoroquinolone-resistance rate among the Campylobacter spp. isolated from humans between 1999 and 2002, from the same hospital laboratory. The use of different methodologies in their study and the current study prevents the direct comparison of the results. However, the possibility of a considerable rise in fluoroquinolone resistance in the 2 years that followed their report cannot be excluded, and should be proven by further monitoring of its incidence.
A total of 31 (75.6 %) of the 41 strains tested were serotypable, representing 10 serogroups. Serotype complex group HS4,13,16,43,50 was the most frequently encountered (22 %), followed by serotype HS2 (14.6 %); both are commonly encountered groups at other geographical locations also (Patton et al., 1991; Nielsen et al., 1997; Rautelin & Hänninen, 1999; Wareing et al., 2002) (Fig. 1
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). Two PFGE groups (P3 and P17) contained two, a further two types (P1 and P4) contained four, and a single type (P7) consisted of eight isolates. Of the 23 RFLP types (R1–R23), 15 were represented by single isolates only. Combining the results of the two typing methods, strains could be assigned to 30 distinct molecular types (MTs) (MT1–30). It is noteworthy that the eight isolates with identical PFGE patterns (P7) also exhibited identical RFLP patterns (R13) and formed a homogeneous MT (MT1) representing 19.5 % of the total, and 22.8 % of the ciprofloxacin-resistant strains (Fig. 1).
Similar to the results of others (Aarts et al., 1995; Rautelin & Hänninen, 1999; Hänninen et al., 2001), we found that the overlap between the results of sero- and molecular typing was not complete. Identical serogroups were encountered in different P and R groups (e.g. HS2 in P4, P11, P12, P14 and P15, or in R6, R8, R10, R16 and R17), while different serogroups could be detected within the same molecular groups (HS2 and HS1,44 in P4, or HS10 and HS1,44 and HS4,13,16,43,50 in R7). On the other hand, six of the eight strains in the cluster MT1 belonged to serogroup HS4,13,16,43,50, while two isolates were non-typable (Fig. 1). The six strains sensitive to fluoroquinolones exhibited no clustering by either of the molecular typing methods or by serotyping (Fig. 1).
Molecular basis of fluoroquinolone resistance
All quinolone-resistant strains, but none of the sensitive ones, were positive by MAMA PCR, suggesting the presence of the Thr-86 to Ile mutation in the QRDR of their gyrA genes. The nucleotide sequence of the gyrA gene between nucleotides 64 and 654 (codons 22 and 218) confirmed the presence of the Thr-86 to Ile replacement in all fluoroquinolone-resistant isolates as the only amino acid change within the QRDR (i.e. between codons 69 and 120). These data, together with those recently published from Kuwait (Albert et al., 2005), show that, similar to other geographical locations (Zirnstein et al., 1999; Engberg et al., 2001; Hakanen et al., 2002; Dionisi et al., 2004; Beckmann et al., 2004), this mutation is the one most commonly encountered in fluoroquinolone-resistant isolates in the Middle East. Extensive polymorphism of the gyrA gene in C. jejuni within as well as outside the QRDR is well documented (Dionisi et al., 2004; Beckmann et al., 2004; Hakanen et al., 2002; Piddock et al., 2003). Based on partial sequences, the 35 fluoroquinolone-resistant strains encountered in this study carried 10 allelic variants of the gyrA gene (G1–G10), although distributed disproportionately (Fig. 1, Table 1). Allelic types G1 and G3 were carried by 10 isolates. A further two groups (G6 and G10) contained three strains, three groups (G2, G5 and G8) consisted of two strains, and three allelic variants (G4, G7 and G9) were represented by single isolates only. Surveying GenBank, only nucleotide sequences identical to allelic variants G3 and G10 were found (GenBank accession nos AJ567826.1 and AJ567825.1, respectively) (Table 1).
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), are closely related. Clonal complexes can strongly associate with certain animal hosts, e.g. poultry (Manning et al., 2003), a commonly consumed food in the Middle East. These reservoirs, in turn, may serve as sources of human infection with strains exhibiting similar or identical geno- and phenotypes. On the other hand, stable C. jejuni clones isolated over an extended period of time from apparently independent sources have also been described (Manning et al., 2001, 2003). The lack of epidemiological data and information on the type distribution of local C. jejuni strains isolated from food and from potential animal reservoirs prevents us from clarifying the nature and epidemiological implications of this uniform cluster of strains within the framework of the current study. Nevertheless, their true clonal nature should be proven by sequence analysis of multiple loci of their genomes (Manning et al., 2003).
Two further strains (#5171 and #102) carried the same allele of the gyrA gene as members of genotype group MT1, one of them belonged to serogroup complex HS4,13,16,43,50, while the other was non-typable (Fig. 1). However, these two isolates exhibited <90 % similarity by PFGE and <70 % similarity by PCR-RFLP to the other members of this group. The other large group of strains with an identical allele of the gyrA gene (G3) exhibited more extensive variations with the different typing techniques. They belonged to four different serogroups, and three isolates were non-typable. Furthermore, none of the isolates in this group exhibited similarities exceeding 85 % by PFGE and 80 % by PCR-RFLP to each other, as can be seen in Fig. 1.
Our data show a high incidence (85.4 %) of fluoroquinolone resistance among C. jejuni strains isolated in Al Ain, UAE. This observation is not biased by the fact that eight, i.e. 22.8 %, of the ciprofloxacin-resistant strains represented a single MT isolated over a considerable period of time. Even if members of MT1 had been counted as only one isolate, the incidence of fluoroquinolone resistance would still be 82.3 %, a very high figure by any comparison. This study, the first of this kind from the south-eastern part of the Arabian Peninsula, was too limited in scale to extrapolate the results to the whole UAE. Nevertheless, since some parts of the Middle East are increasingly frequented travel destinations, as well as relatively high-risk areas to acquire campylobacter infections (Ekdahl & Andersson, 2004), the data should stress the need for the establishment of an effective antibiotic surveillance in the region. Furthermore, they also indicate that alternatives to fluoroquinolones for the treatment of fairly severe diarrhoeal cases with unconfirmed aetiology should be sought.
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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