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Serichantalergs eta/. Gut Pathogens 2010, 2:15
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RESEARCH Open Access
PFGE, Liar serotype, and antimicrobial resistance patterns among Campylobacter jejuni isolated from travelers and US military personnel with acute diarrhea in Thailand, 1998-2003 Oralak Serichantalergs 1.2·. Piyarat Pootong 1, Anders Dalsgaard2
, Ladaporn Bodhidatta 1, Patricia Guerrl,
David R Tribble4, Sinn Anuras5
, Carl J Mason 1
Abstract
Background: Compylobocrer 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 31 2 C. jejuni isolates were obtained from travelers (n = 46) and U.S. mi litary 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, respeaively. From 2001 -2003, resistance to tetracycline (8 1.go,,), trimethoprimsulfamethoxazole (57.9%), ampicillin (28.9%), kanamycin (5.9%), sulfisoxazole (3.9%), neomycin (2.0%), and streptomycin (0.7%) was obseNed. Combined PFGE analysis showed considerable genetic diversity among the C. jejuni isolates; however, four PFGE clusters included isolates from the major Liar serotypes (HL: 36, HL: 1 1, 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 deteaed spatial and temporal C. jejuni clonality responsible for the common sources of Compylobocrer gastroenteritis.
Background Campylobacter jejuni is a major cause of gastroenteritis worldwide, especially in children, travelers, and military personnel deployed to develop ing countri es [1 -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. jeju11i and C. coli have been developed [8- 10]. Molecu lar
• Correspondence: [email protected] 1 Department of Enteroc D1seases. Armed Forces Research lnsmute of Med1cal Sciences, 315/6 RaJVIthl Road. Phayathai, Bangkok 10400. Thailand Full hst of author information is available at the end of the amcle
techniques such as RF LP, RAPD, PFGE, AFLP, and MLST have also been applied to C. jejwri 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. jejttni 112,13]. 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 [14-16]. Furthermore, the combination of PFGE and other typing techniques can identify
() BioMed Central C 2010 Se1ichantalergs et al. hcensee 81oMed Central Ltd. Th1s1s an Open Access arude d1st11buted under the teuns of the C1eative Commons Attllbuuon lJcense lhttp1/creativecommons.org/llccnses/by/2.0). whiCh pe1m1t.s UIIICitllcted use. dlstllb!Jtlon. and reproduction 1n any med1um, prOVIded the 01191nal w01k is propeliy Cited
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4. TITLE AND SUBTITLE PFGE, Liar serotype, and antimicrobial resistance patterns amongCampylobacter jejuni isolated from travelers and US military personnelwith acute diarrhea in Thailand, 1998-2003
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14. ABSTRACT Background: Compylobocter jejuni is a major cause of gastroenteritis worldwide. In Thailand, severalstrains 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 inThailand from 1998-2003, strains of C. jejuni were isolated and phenotypically identified, serotyped, testedfor 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 ciprofioxacin resistance wasobserved in 94.9% and 93.0% of the isolates, respectively. From 2001 -2003, resistance to tetracycl ine (81.9%), trimethoprimsulfamethoxazole (57.9%), ampicillin (28.9%), kanamycin (5.9%), sulfisoxazole(3.9%), neomycin (2.0%), and streptomycin (0.7%) was obseNed. Combined PFGE analysis showedconsiderable genetic diversity among the C. jejuni isolates; however, four PFGE clusters included isolatesfrom the major Lior serotypes (HL: 36, HL: 11 , HL: 5 and HL: 28). The PFGE analysis linked individualC. jejuni clones that were obtained at U.S. military exercises with specific antimicrobial resistancepatterns. Conclusions: In summary, most human C. jejuni isolates from Thailand were multi-resistant toquinolones and tetracycline. PFGE detected spatial and temporal C. jejuni clonality responsible for thecommon sources of Compylobocter gastroenteritis.
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Serichantalergs eta/. Gut Pathogens 2010, 2:15 http://www.gutpathogens.com/content/2/111 5
common sources of Campylobacter and other bacterial infections [17-19].
In Thailand, C. jejuni was isolated and identified as a major diarrheal pathogen among children and adult travelers, including U.S. military personnel [20-23]. 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
Page 2 of 11
common, and serotypes HL: 9 and HL: 2 were less common. The majority of C. jejuni isolates from serotype HL: 5 (11/23) were isolated in 2001, and most C. jejuni HL: 19 isolates ( 15/17) were obtained in 1999. The untypable isolates comprised 54.3% (25/46) of the isolates from foreign travelers in 2001-2002 compared to 11.3% (30/266) of the isolates from U.S. military personnel in this study. The adult travelers from Bumrungrad Hospital spanned several nationalities, including Japanese, European, American, and Australian. The high percentage of untypable isolates might reflect the diverse foods consumed by these travelers, while the low percentage of untypable isolates in the military personnel might reflect the limited diet consumed by U.S. military personnel on deployment.
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 susceptibility tests In 1998-2003, 94.9% (296/312) and 93.0% (289/311) of the isolates were resistant to quinolones (NAL and CIP), but 99.0% (306/309) were susceptible to macrolides (ERY and AZM). The high prevalence of quinolone (NAL and CIP) resistance and macrolide (ERY and AZM) sensitivity is consistent with previously reported results from Thailand [21,24,26]. For the 138 C. jejuni isolates from 2001-2003, the percentages of resistant isolates detected were as follows (Table 3): CF, 100% (138/ 138); TE, 81.9% (113/138); SXT, 58.0% (80/138); AMP, 30.4% (42/138); KM, 6.5% (9/138); SU, 3.6% (5/138); NM, 2.2% (3/138); and SM, 0.7% (11138). None of these 138 isolates were resistant to GM, CM or CL. Notably, tetracycline resistance was also detected in over 80% of
Table 1 Numbers of C. jejuni isolates and two most common Lior serotypes by study location/year
Year Study location Enrolled diarrhea cases Number of C. jejunl isolates Two most common Uor serotypes
(Total = 1292) (Total = 312) (Number of isolates)
1998 Kanjanaburi 166 10 HL: 11 (3), HL: 36 (2)
Cholburi 12 HL: 36 (3), HL: 11 (2)
1999 Korat 198 84 HL: 36 (33), HL: 19 (15)
2000 Nakornsrithammarat 256 68 HL: 11 (23), HL: 36 (19)
2001 Phitsanulok 153 54 HL: 5 (11), HL: 36 (8)
2001-2002 Bangkok 417 46 untypable (35), HL:11 (5)
2002 Sakaew 54 15 HL: 36 (7), untypable (4)
2003 Pranburi 48 23 HL: 4 (7), HL: 36 (3)
Serichantalergs et al. Gut Pathogens 2010, 2:15 http://www.gutpathogens.com/content/2/1/15
Page 3 of 11
Table 2 Lior serotype distribution of C. jejuni isolates among foreign travelers and U.S. military personnel (1998-2003)
Uor serotype Serotypes In foreign travelers % (n) Serotypes In U.S. military personnel % (n) %Total (n)
(Total= 46) (Total= 266) (Total= 312)
HL: 36 6.5 (3) 28.2 (75) 25.0 (78)
HL: untypable 54.3 (25) 11.7 (30) 17.6 (55)
HL: 11 6.5 (3) 14.7 (40) 13.8 (43)
HL: 5 4.3 (2) 7.9 (21) 7.4 (23)
HL: 1 0.0 7.5 (19) 6.1 (19)
HL:4 4.3 (2) 5.6 (15) 5.4 (17)
HL: 19 0.0 6.0 (17) 5.4 (17)
HL: 7 2.2 (1) 4.9 (13) 4.5 (14)
HL: 114 4.3 (2) 4.1 (10) 3.8 (12)
HL: 28 4.3 (2) 3.8 (10) 3.8 (12)
HL: 102 2.2 (1) 1.9 (5) 1.9 (6)
HL: 2 0.0 1.9 (5) 1.6 (5)
HL:9 0.0 1.5 (4) 1.3 (4)
HL: 17 4.3 (2) 0.4(1) 1.0 (3)
HL: 41 2.2 (1) 0.0 0.3 (1)
HL:53 0.0 0.4 (1) 0.3 (1)
HL:6 2.2 (1) 0.0
HL:86 2.2 (1) 0.0
isolates that were similar to C. jejuni isolates from Taiwan (95%) [27], Korea (87%) [6,28], Canada, and the U.S. (SO%) [29,30).
The two most common resistance patterns observed in these 138 isolates were multiple resistance to four antimicrobials (NAL, CIP, CF, and TE), observed in 79.0% (109/138) of the isolates, and multiple resistance to five antimicrobials (NAL, CIP, CF, TE, and SXT), detected in 47.8% (66/138) of the isolates. Another resistance pattern (NAL, CIP, CF, TE, SXT, and AMP) was found in 15.9% (22/138) of the isolates, and a fourth pattern of resistance (NAL, CIP, CF, TE, and KM) was detected in 5.8% (8/138) of the isolates. The first common pattern was similar to a previous report of antimicrobial resistance to NAL, CIP, and TE in 53% of clinical C. jejuni isolates in Thailand [31]. These results confirm widespread quinolone and tetracycline resistance among C. jejuni isolates from traveler's diarrhea in Thailand.
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
0.3 (1)
0.3 (1)
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. [32-35]. 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].
PFGE and antimicrobial resistance by study location Dendrograms A thru D in Figs. 2, 3, 4, and 5 were generated for four study locations from 2001-2003. Dendrogram A includes 46 isolates obtained from Bumrungrad Hospital, Bangkok, over the two-year period from 2001-2002 (Figure 2). Although the common resistance pattern (NAL, CIP, CF, and TE) was observed in 76.1%
Serichantalergs er at. Gut Pathogens 2010, 2:15
hnpJ/www.gutpathogens.com/content/2/ 1/ 15
% Similarity
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Serotype {n) Total isolates HL: 36(8), HL: Untype(3) 11 H\.: :16(1) Ut.; 7( l), llW Unt~l)
HL: 2{2), HL: Unlype(t) HL: Untype:(1) HL: Unt,-pr~(1)
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HL: 1(2) HL: 4(4), HL : Untype(2)
UL: ol(5) UL: 4(2), HL:Jtl(t) HL: 4(1) Hl : 36(1) til : 36(1). HL: Unty~l) HL:4(1)
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ttl.: U411 ) Ill.: 2(1), HL:Untype ( l) Hl.;fC II H\..;t(f),Kl: Uroryp.fll HL:~ tt. Hl.:1Jneype(t) H\.:U~ttyp.(U HL: ~~~ HL: UI"CnN'( t) HL:S)(I .. HL.;~ HL.:If\11 Hl.: U""YP"I II HL: 7( 1) HL:U~Itl
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Figure 1 Dendrogram and Lior serotype of 312 C. jejuni isolates f rom travelers' diarrhea in Thailand (1998-2003). PFGE cluster analysis
of a Smai/Kpnl restriction enzyme digest of genomic DNA from 312 C. jejuni isolates analyzed at an 80% similarity cut off, resulting in 62
genotypes distributed between Uor serotypes.
Serichantalergs et al. Gut Pathogens 2010, 2:15 http://www.gutpathogens.com/content/2/1/1 5
Page 5 of 11
Table 3 Antimicrobial resistance among 138 human C. jejuni isolates from Thailand (2001-2003)
Antimicrobial Percentage of resistant isolates (number of isolates)
Phitsanulok 2001 Bangkok 2001-2002 Sakaew 2002 Pranburi 2003 p-value • (n =54) (n = 46) (n = 15) (n = 23)
Nalidixic acid 96.2% {52} 93.5% {43} 86.6% {13} 100.0% {23} NS Ciprofloxacin 96.2% {52} 87.0% {40} 86.6% {13} 91.3% {21} NS Erythromycin 0.0%{0} 6.5% {3} 0.0% {0} 0.0%{0} NS Azithromycin 0.0%{0} 6.5% {3} 0.0% {0} 0.0%(0} NS Tetracycline 90.7% {49} 76.0% {35} 86.6% {13} 69.5% {16} NS
Trimethoprim- 90.7% {49} 58.7% {27} 26.7% {4} 0.0%{0} <0.001 sulfamethoxazole
Ampicillin 31.5% {17} 30.4% {14} 60.0% {9} 8.7% {2} <0.05
Kanamycin 0.0%{0} 2.2% {1} 20.0% {3} 21.8% {5} <0.001
Sulfisoxazole 0.0% {0} 10.9% {5} 0.0% {0} 0.0%(0} NA
Neomycin 0.0%{0} 2.2% {1} 1.7% (1} 1.7% {1} NA
Streptomycin 0.0% {0} 0.0% {0) 1.7% {1} 0.0%{0} NA
Gentamicin 0.0% {0} 0.0% {0) 0.0% {0} 0.0% {0} NA
Colistin 0.0%{0} 0.0% {0) 0.0% {0} 0.0% {0} NA
Chloramphenicol 0.0%{0} 0.0% {0} 0.0%{0} 0.0% {0} NA
Cephalothin 100.0% {52} 100.0% {60} 100.0% {15} 100.0% {23} NA
• Pearson chi-square Monte Carlo (two-tailed); NS, non-significant; NA, not applicable.
(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 toNAL, 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 D 1 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 D 1 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
Serichantalergs eta/. Gut Pathogens 2010, 2:15
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R R R R
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Figure 2 Dendrog ram of C. jejuni isolat es with Lior serotypes and antimicrobial resistance from Bumrungrad Hospital (2001·2002).
Dendrogram A: PFGE cluster analysis of a Smai/Kpnl restriction enzyme digest of genomic DNA from 46 C. je)uni isolates w ith marked clonal
heterogeneity, unrelated antimicrobial resistance patterns. and 25 untypable isolates from this study si te. R. resistance; I, intermediate
susceptibility; 5, susceptible; NO, not tested.
Serichantalergs et al. Gut Pathogens 2010, 2:15 http://www.gutpathogens.com/content/2/ 1/ 1 5
Page 7 of 11
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I I I till il Untypable S R R S S S S S S S S R R R S
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Figure 3 Dendrogram of 54 C. j ej uni isolates w ith Lior serotypes and antimicrobial resistance from Phitsanulok 2001 . Dendrogram B:
PFGE cluster analys1s of a SmaVKpnl restriction enzyme digest of genomic DNA from 54 C. JeJuni ISolates from the Cobra Gold exercises
(Phitsanulok. 2001) with evidence of clonality due to similarity in the PFGE patterns. Uor serotype(s) and related specific antimicrobial
susceptibility pan ems (genotypes B I and B3a). R. resistance; I, intermediate susceptibility; S. susceptible.
Serichantalergs eta/. Gut Pathogens 2010, 2:1 5 http:/ /www.gutpathogens.com/content/2/1 /15
Similarity Smal
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Page 8 of 11
Dendrogram C
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1 ' I !Ill• til Unlypable S R R S S S S S S S S R R S S I I I IIIII I I HL:2 S R R S R S S S S S S R R R S
.I l l I I I I HL:114 S R R S R S S R S S R R R S S I. I II II lfl Untypable S R R S S S S S S S S R S S S I I II I II I I Untypable S S S S R S S S S S S R S R S
Figure 4 Dendrogram of 15 C jejuni isolates w ith Liar sera types and ant imicrobial resistance from Sakaew 2002. Dendrogram C: PFGE cluster analysis of a Smai!Kpnl restriaion enzyme digest of genomic DNA of 15 C. jejuni isolates from the Cobra Gold exercises (Sakaew. 2002} with evidence of clonality due to similarity in the PFGE patterns (>99.2%), identical Lior serotype (HL:36} and a specific antimicrobial susceptibility pattern (genotype C 1 with ampicillin resistance). R, resistance; 5, susceptible.
over a 2-year period, a simi lar 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.
Methods Sources of isolates Under approved human use protocols, stool specimens were obtained fro m patients with d iarrhea 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.
Dendrogram D
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I I II I I 1111 HL: I s R R s s s s s s s s R R s s tt I Iii • Jil !J~ Ill HL:36 s R R s s s s s s s s R R s s
Figure 5 Dendrogram of 23 C. jejuni isolates with Liar serotypes and antimicrobial resistance from Pranburi 2003. Dendrogram D: PFGE cluster analysis of a Smai!Kpnl restriaion enzyme digest of genomic DNA of 23 C. jejuni isolates from the Cobra Gold exercises (Pranburi, 2003) with evidence of clonality due to similarity in the PFGE patterns (I 00%}, identical Lior serotype (HL: 4} and specific antimicrobial susceptibility patterns (genotype Dl with additional kanamycin resistance). R. resistance; I, intermediate susceptibility; 5, suscept ible.
Serichantalergs et al. Gut Pathogens 2010, 2:15 http://www.gutpathogens.com/content/2/1/15
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, H2S TSI, oxygen tolerance, and microaerobic growth at 25oC, 37oC, 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 -70oC 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], with modifications. BAP subcultures of patient isolates at 18- to 48-h were suspended in Mueller Hinton broth (BD Diagnostic Systems, Sparks, MD, USA.) to obtain a turbidity equivalent to a 1.0 McFarland standard, and suspensions were inoculated onto Mueller Hinton II agar supplemented with 5% sheep blood. At the time of each study, all C. jejuni isolates were tested for susceptibility to the following antimicrobials (BD Diagnostic Systems): NAL (30 ~g), CIP (5 ~g), ERY (15 ~g), and AZM (15 ~g). The 138 C. jejuni isolates in 2001-2003 were further tested for susceptibility to 11 additional antimicrobials by disk diffusion assay. These antimicrobial disks included AMP (10 ~g), CM (30 ~g), KM (30 ~g), GM (10 ~g), SM (10 ~g), TE (30 ~g), SXT (1.25/23.75 ~g), SU (250 ~g), CL (10 ~g), NM (30 ~g), and CF (30 ~g). Disks were placed on the surfaces of inoculated Mueller Hinton II agar plates. Inoculated plates were incubated at 37oC for 24 h in a microaerobic environment The plates were re-incubated up to 48 h if insufficient growth of C. jejuni isolates on
Page 9 of 11
the Muller Hinton II agar plates was obtained at 24 h. Because no standardized interpretive criteria exist for Campylobacter spp., the inhibition zone diameters were measured and interpreted following the disk manufacturer's instructions and compared against the Clinical and Laboratory Standards Institute (formerly NCCLS) standard guidelines for aerobic gram-negative bacilli to interpret the results as susceptible, intermediate, or resistant [43]. Escherichia coli ATCC 25922 and Staphylococcus au reus A TCC 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 following abbreviations were used: RFLP: restriction fragment length polymorphism; RAPD: random amplification of polymorphic DNA; PFGE: pulsed-field gel electrophoresis; AFLP: amplified fragment length polymorphism; MLST: multilocus sequence typing; MIC: minimal inhibitory concentration; HL: heat-labile; NAL: nalidixic acid; CIP: ciprofloxacin; ERY: erythromycin; AZM: azithromycin; AMP: ampicillin; CM: chloramphenicol; KM: kanamycin; GM: gentamicin; SM: streptomycin; TE: tetracycline; SXT: trimethoprim·sulfamethoxazole; SU: sulfisoxazole; CL: colistin; NM: neomycin; CF: cephalothin.
Acknowledgements All of the study projects described herein were supported financially by the Military Infectious Diseases Research Program, United States Army Medical Research and Materiel Command in Fort Detrick. MD, USA. We are grateful to the National Laboratory for Enteric Pathogens in Winnipeg, Manitoba, Canada for providing antiserum to perform the Lior serotyping. The views expressed here are those of the authors and are not to be construed as reflecting the views of the United States Department of Defense or the United States Army.
Serichantalergs eta/. Gut Pathogens 2010, 2:15 http://www.gutpathogens.com/content/2/1/15
Author details 1 Department of Enteric Diseases, Armed Forces Research Institute of Medical
~ciences, 315/6 Rajvit~i Road, Pha~athai, Bangkok 10400, Thailand. Department of Vetennary Pathob1ology, Faculty of Life Sciences, ~openhagen University, Stigboejlen 4, DK-1870 Frederiksberg C. Denmark. Naval Medical Research Center, 503 Robert Grant Ave, Silver Spring, MD
20910, USA 4Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-5119, USA 5Bumrungrad Hospital. 33 Sukhumvit Sci 3, Bangkok 10110. Thailand.
Authors' contributions OS designed and carried out the study project (including the data analysis and preparation of the draft manuscript), PP performed the PFGE of the C. jejuni isolates, AD provided ideas and comments on the draft manuscript, LB
analyzed the epidemiological data for the C. jejuni isolates, PG provided expertise on the molecular biology of C. jejuni, DT directed the patient recruitment in the US military exercise. SA supported the study project etiology of acute diarrhea at Bumrungrad Hospital. and OM conceived the idea for the study (and performed statistical analysis and worked on the final manuscript). All authors read and approved the final manuscript.
Competing Interests The authors declare that they have no competing interests.
Received: 4 October 2010 Accepted: 10 November 2010 Published: 10 November 2010
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doi:10.1186/1757-4749-2-1 s Cite this article as: Serichantalergs et a/.: PFGE. Uor serotype, and antimicrobial resistance patterns among Campylobader jejuni isolated from travelers and US military personnel with acute diarrhea in Thailand, 1998-2003. Gut Pathogens 2010 2:15.
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