PFGE, Lior serotype, and antimicrobial resistance patterns among Campylobacter jejuni isolated from travelers and US military personnel with acute diarrhea in Thailand, 1998-2003

Background Campylobacter jejuni is a major cause of gastroenteritis worldwide. In Thailand, several strains of C. jejuni have been isolated and identified as major diarrheal pathogens among adult travelers. To study the epidemiology of C. jejuni in adult travelers and U.S. military personnel with acute diarrhea in Thailand from 1998-2003, strains of C. jejuni were isolated and phenotypically identified, serotyped, tested for antimicrobial susceptibility, and characterized using pulsed-field gel electrophoresis (PFGE). Results A total of 312 C. jejuni isolates were obtained from travelers (n = 46) and U.S. military personnel (n = 266) in Thailand who were experiencing acute diarrhea. Nalidixic acid and ciprofloxacin resistance was observed in 94.9% and 93.0% of the isolates, respectively. From 2001-2003, resistance to tetracycline (81.9%), trimethoprim-sulfamethoxazole (57.9%), ampicillin (28.9%), kanamycin (5.9%), sulfisoxazole (3.9%), neomycin (2.0%), and streptomycin (0.7%) was observed. Combined PFGE analysis showed considerable genetic diversity among the C. jejuni isolates; however, four PFGE clusters included isolates from the major Lior serotypes (HL: 36, HL: 11, HL: 5, and HL: 28). The PFGE analysis linked individual C. jejuni clones that were obtained at U.S. military exercises with specific antimicrobial resistance patterns. Conclusions In summary, most human C. jejuni isolates from Thailand were multi-resistant to quinolones and tetracycline. PFGE detected spatial and temporal C. jejuni clonality responsible for the common sources of Campylobacter gastroenteritis.


Background
Campylobacter jejuni is a major cause of gastroenteritis worldwide, especially in children, travelers, and military personnel deployed to developing countries [1][2][3][4]. In recent years, a high prevalence of infection and an increased resistance to the antimicrobials used to treat diarrhea have been documented [5,6]. C. jejuni and C. coli can be phenotypically characterized by growth characteristics, biochemical reactions, and hippurate hydrolysis [7]. Serotyping techniques for C. jejuni and C. coli have been developed [8][9][10]. Molecular techniques such as RFLP, RAPD, PFGE, AFLP, and MLST have also been applied to C. jejuni isolate characterization [11].
PFGE is a well-known technique standardized by the Centers for Disease Control and Prevention (CDC) for subtyping Salmonella spp., Shigella spp., and Vibrio spp., in addition to C. jejuni [12,13]. Unlike other enteric bacteria, Campylobacter is a genetically diverse organism that undergoes intra-and inter-genomic exchange. However, PFGE is considered to be the most discriminatory method of characterizing C. jejuni and C. coli and identifying specific Campylobacter spp. in outbreak studies [14][15][16]. Furthermore, the combination of PFGE and other typing techniques can identify common sources of Campylobacter and other bacterial infections [17][18][19].
In Thailand, C. jejuni was isolated and identified as a major diarrheal pathogen among children and adult travelers, including U.S. military personnel [20][21][22][23]. A high prevalence of infection with fluoroquinolone-resistant C. jejuni was previously reported in both Thai children and U.S. military personnel [3,24]. However, epidemiological data on Campylobacter infection among travelers and expatriates as well as their susceptibility to other antimicrobials have not been described in Thailand in several years. This prompted us to investigate and characterize the C. jejuni isolates responsible for gastroenteritis in adult travelers by combining antimicrobial resistance data, serotype classification, and PFGE.

Results and Discussion
Frequency and distribution of C. jejuni serotypes From a total of 312 C. jejuni isolates obtained in this study, 266 isolates were from U.S. soldiers and 46 were from foreign travelers seen at Bumrungrad Hospital, Bangkok. The prevalence of C. jejuni from diarrheal cases was detected as 11.0% (46/417) in the Bumrungrad Hospital study and 30.4% (266/875) in samples from U.S. military personnel in the Cobra Gold exercises of 1998-2003 (Table 1). Overall, a total of 16 Lior serotypes were detected; the three most common serotypes were HL: 36, HL: 11, and HL: 5; these serotypes accounted for 25.0% (78/312), 13.8% (43/312), and 7.4% (23/312) of all of the isolates, respectively. Untypable strains composed 17.6% (55/312) of the isolates. The distribution of the serotypes and the incidence of C. jejuni isolates among foreign travelers and U.S. military personnel are shown in Table 2. The two most common serotypes by study location and year are shown in Table 1.
The incidence of the serotypes described herein were included from the first to tenth ranks among the global isolates [25] but were slightly different from those reported previously in Thai children [24]. For example, serotype HL: 5 was detected infrequently among Thai children [24]. In this study, serotype HL: 5 was more common, and serotypes HL: 9 and HL: 2 were less common. The majority of C. jejuni isolates from serotype HL: 5 (11/23)

PFGE analysis of C. jejuni isolates
At an 80% similarity level, a dendrogram combining data for all 312 C. jejuni isolates from Thailand was clustered into 62 genotypes ( Figure 1). Four major genotypes composed 49.7% (155/312) of the C. jejuni isolates in this study, and 30 of the 62 genotypes included only a single C. jejuni isolate. Of the 55 isolates that were untypable by Lior serotyping, 42 could be grouped into a genotype with other known serotypes.
Antimicrobial resistance by study location and the results of the chi-square test are shown in Table 3. Interestingly, KM-resistant isolates were detected in 6.5% (9/138) of the isolates, but there was a significant difference (from 2.2% to 21.8%) in the frequency of resistance in isolates from different locations (p < 0.001). The percentage of AMP-resistant isolates also varied by location (p < 0.05). The resistance of isolates to SXT varied greatly from 90.7% at Phitsanulok to 58.7% at Bumrungrad Hospital, 26.7% at Sakaew, and to undetectable levels in isolates from Pranburi (p < 0.001). The finding of differences in AMP, KM, and SXT resistance among C. jejuni isolates from selected sites should not be considered as indicative of significant changes over time in Thailand.
In this study, the antimicrobial susceptibility tests were performed by disk diffusion assay. Other methods, including agar dilution and broth micro dilution methods, and epsilometer test (E-tests) have been used by different laboratories to measure antimicrobial susceptibilities for Campylobacter spp. [32][33][34][35]. However, good agreement of antimicrobial susceptibility test between disk diffusion and agar dilution tests has been observed in several classes of antimicrobials especially quinolone/ fluoroquinolones and aminoglycosides suggesting that disk diffusion test could be used as qualitative assay but not quantitative assay for antimicrobial susceptibility among Campylobacter spp. [36]. Although another study suggested that interpretation of erythromycin by disk diffusion assay was unreliable and should be confirmed by MIC-based methods [37], but our previous data showed high correlation of antimicrobial susceptibility by disk diffusion and agar dilution tests in erythromycin and azithromycin-resistant C. jejuni and C. coli isolates [38].   (35/46) of the isolates, there was substantial heterogeneity in the PFGE patterns ( Figure 2). The PFGE analysis suggests diverse genotypes. No specific multi-resistant antimicrobial patterns were noted in the remainder of these isolates. The dendrogram and the diversity of serotypes and antimicrobial susceptibility patterns suggest that these isolates are heterogeneous and clonally diverse. Dendrogram B includes the 54 isolates collected from U.S. soldiers in Phitsanulok in 2001 ( Figure 3). As above, 87% (47/54) of these isolates were generally multi-resistant to NAL, CIP, CF, and TE; in addition, 87% (47/54) were resistant to NAL, CIP, CF, and SXT. The genotype B3 included 24 isolates exhibiting 86.1% similarity that belonged to three serotypes: HL: 5 (11), HL: 102 (3), and HL: untypable (10). A subset of 12 isolates (B3a) within the genotype B3 had 99.7% similarity and resistance to AMP, suggesting clonality ( Figure 3). Similarly, genotype B1 consisted of five isolates of serotype HL: 36 with 100% similarity and an identical antimicrobial resistance pattern (NAL, CIP, CF, TE, and SXT). C. jejuni isolates in genotypes B1 and B3a were isolated over a 5-day period and 8-day period, respectively.
Dendrogram C (Figure 4) shows 15 isolates collected from U.S. soldiers in Sakaew over a 1-month period in 2002. Genotype C1 includes 5 isolates of serotype HL: 36 with 99.2% similarity and an identical antimicrobial resistance pattern (NAL, CIP, CF, AMP, and TE), which also suggests clonality. The other 10 isolates had <67.5% similarity and included five serotypes and seven antimicrobial resistance patterns, suggesting more diverse sources ( Figure 4). All C. jejuni isolates in genotype C1 were isolated over a period of 6 days.
Dendrogram D ( Figure 5) shows the 23 isolates collected from U.S. soldiers in Pranburi during a 1-month exercise in 2003 and genotype D1 includes a unique cluster of five serotype HL: 4 isolates with an unusual multi-resistant antimicrobial pattern (NAL, CIP, CF, KM, and TE), suggesting clonality for these isolates. Similar to dendrograms B and C, genotype D1 included C. jejuni isolates collected during a 10-day period.

Conclusions
In summary, our study demonstrates the usefulness of PFGE in local epidemiological studies or in the study of small outbreaks occurring over a short time interval rather than in the long-term epidemiological studies that have been studied by others [26]. This study confirmed the existence of common C. jejuni clones that are associated with specific serotypes and multiple antimicrobial resistance patterns in the Cobra Gold military exercises but not in the traveler's diarrhea study at Bumrungrad Hospital. A possible explanation for these findings is that the Cobra Gold military exercises took place at particular locations with short durations (1 month). Diarrhea cases among soldiers might also be expected to be caused by common exposures. Our finding of an association between PFGE and serotype with the antimicrobial resistance patterns in these exercises differed from other studies in which the correlation between PFGE and multi-antimicrobial resistance was low [28,39,40]. In the Bumrungrad Hospital study, where diarrhea cases occurred in diverse populations   over a 2-year period, a similar low correlation was observed between serotype and antimicrobial resistance. Our data suggest that these patients became infected with unrelated C. jejuni isolates. Comprehensive monitoring of human C. jejuni isolates, including animal and environmental sources, should be expanded in Thailand to monitor antimicrobial resistance and to better document potential sources of infection.

Sources of isolates
Under approved human use protocols, stool specimens were obtained from patients with diarrhea and from asymptomatic controls in an acute diarrhea study among foreign travelers from highly developed countries at Bumrungrad Hospital in Bangkok, Thailand during 2001-2002. Stool specimens were collected from U.S.  soldiers with acute diarrhea and from asymptomatic controls; the soldiers were deployed for the Cobra Gold exercises lasting one for four weeks at different sites in Thailand during 1998-2003. Only C. jejuni isolates from acute diarrhea cases were included in this study. Table 1 describes the number of C. jejuni isolates from cases in each study location.

Isolation and identification
All stool specimens were cultured for Campylobacter spp. using a modified filtration method described previously [41]. Suspected colonies, growing on Brucella Agar (Difco, Detroit, MI, USA) with 5% sheep blood (BAP), were identified as Campylobacter spp. by colony characteristics, Gram staining, oxidase tests, and catalase tests, followed by phenotypic tests including hippurate hydrolysis, nitrate reduction, H 2 S TSI, oxygen tolerance, and microaerobic growth at 25°C, 37°C, and 42°C. C. jejuni isolates were differentiated from C. coli by the hippurate hydrolysis test. All C. jejuni isolates were kept in glycerol medium at -70°C for further analysis.

Serotyping
Lior serotyping was performed by an agglutination assay with specific antiserum obtained from the National Laboratory for Enteric Pathogens (NLEP) in Winnipeg, Manitoba, Canada. These antisera were routinely used to serotype C. jejuni and C. coli isolates at AFRIMS. The antisera detect heat-labile antigens [9] and identify 33 common HL serotypes.
Antimicrobial susceptibility testing C. jejuni isolates were tested for susceptibility to antimicrobial drugs using a disk diffusion assay as described previously [42] [43]. Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as standard organisms for all disk diffusion assays.

Statistical analysis
Pearson's chi-square tests of independence for the antimicrobial susceptibility data (NAL, CIP, ERY, AZM, TE, SXT, AMP, and KM) between locations were performed using the Monte Carlo-exact (2-sided) method in SPSS version 12.0 (SPSS Inc., Chicago, IL, USA). A p-value < 0.05 was considered significant.

PFGE
PFGE was performed according to the One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping by the CDC [12] with the minor modifications described below. The cell density of each isolate was adjusted to an O.D. of 0.6 using a spectrophotometer (Spectramax 190; Molecular Devices, Sunnyvale, CA, USA) that was different from the spectrophotometers suggested by the CDC. The PFGE patterns were analyzed to generate dendrograms of the combined SmaI and KpnI similarities using BioNumerics version 5.0 (Applied Maths, Sint-Martens-Latem, Belgium) by UPGMA type and Dice coefficient with 1.5% optimization and tolerance. Dendrograms were made for the composite data of all of the C. jejuni isolates and for the different locations.

Abbreviations
The