Antimicrobial Resistance in Salmonella enterica Serovar Typhi Isolates from Bangladesh, Indonesia, Taiwan, and Vietnam
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Chien-Shun Chiou, Tsai-Ling Lauderdale, Dac Cam Phung, Haruo Watanabe, Jung-Che Kuo, Pei-Jen Wang, Yen-Yi Liu, Shiu-Yun Liang and Pei-Chen Chen Antimicrob. Agents Chemother. 2014, 58(11):6501. DOI: 10.1128/AAC.03608-14. Published Ahead of Print 18 August 2014.
Antimicrobial Resistance in Salmonella enterica Serovar Typhi Isolates from Bangladesh, Indonesia, Taiwan, and Vietnam Chien-Shun Chiou,a Tsai-Ling Lauderdale,b Dac Cam Phung,c Haruo Watanabe,d Jung-Che Kuo,a Pei-Jen Wang,a Yen-Yi Liu,a Shiu-Yun Liang,a Pei-Chen Chenb Centers for Disease Control, Taichung, Taiwana; National Health Research Institutes, Zhunan, Taiwanb; National Institute of Hygiene and Epidemiology, Hanoi, Vietnamc; National Institute of Infectious Diseases, Tokyo, Japand
S
almonella enterica subspecies enterica serovar Typhi, the cause of typhoid fever, is transmitted primarily by ingestion of contaminated food and water. It was estimated to cause 21.6 million illnesses and 216,000 deaths during 2000 (1). Despite the fact that the incidence of typhoid fever has declined markedly in developed countries, it remains a major cause of morbidity in less-developed countries. South-central Asia and Southeast Asia are regions with high incidences of typhoid fever (⬎100/100,000 cases/year, respectively). In recent decades, cases of typhoid fever in developed countries have been associated predominantly with travelers returning from areas where typhoid fever is endemic (2, 3). Antimicrobials are the mainstay of therapy for typhoid patients. However, the intensive use of first-line antimicrobials, such as ampicillin, chloramphenicol, and cotrimoxazole, has led to the emergence and global spread of multidrug-resistant (MDR) S. Typhi strains (4). Due to increasing resistance to the antimicrobials used traditionally for therapy, the use of fluoroquinolones, such as ciprofloxacin, for the treatment of typhoid has become more common in developing countries. The use of fluoroquinolones has also led to a rapid increase in reduced susceptibility of S. Typhi to these therapeutics. MDR S. Typhi with reduced ciprofloxacin susceptibility has become common in Africa and South and Southeast Asia (3, 5–7). In recent years, ciprofloxacin-resistant S. Typhi has been reported frequently (8–12). MDR in S. Typhi is almost exclusively associated with selftransmissible IncHI1 plasmids, which carry a panel of genes conferring resistance to several first-line antimicrobials, including ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline, and trimethoprim (13, 14). This plasmid type has also been identified in MDR S. Paratyphi A (15). A study conducted by Holt et al. (16) indicated that prior to 1995, MDR typhoid was caused by a diverse range of S. Typhi and MDR plasmids. However, from 1995 onwards, 98% of MDR S. Typhi isolates were of S. Typhi haplotype H58, carrying the IncHI1 plasmid sequence type 6 (PST6). The PST6 plasmid confers on the host the ability to grow
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in high-salt medium, which may provide a competitive advantage over S. Typhi strains carrying other plasmid types. Currently, the majority of MDR S. Typhi H58 clone isolates are resistant to nalidixic acid, and they are widespread in Africa and Asia (17–20). In this study, we investigated the genetic relatedness and antimicrobial resistance among S. Typhi isolates from four Asian countries: Bangladesh, Indonesia, Taiwan, and Vietnam. The results indicate that the majority of S. Typhi isolates from Bangladesh and Vietnam are MDR and genetically closely related, 40% of isolates from Bangladesh are ciprofloxacin resistant, and several resistance mechanisms in the isolates from Bangladesh may underlie their resistance to nalidixic acid and ciprofloxacin. MATERIALS AND METHODS Bacterial isolates. Of 180 S. Typhi isolates included in this study, 38 were from Bangladesh and were collected in 2007, 55 were from Indonesia and were collected between 2007 and 2009, 36 were from Taiwan and were collected between 2007 and 2009, and 51 were from Vietnam and were collected between 2007 and 2008. The Indonesian isolates were collected in Taiwan by the Centers for Disease Control, Taiwan, from Indonesian migrant workers if the isolates were recovered within 60 days of their arrival to Taiwan and from returning travelers who visited only Indonesia and developed symptoms within 60 days. The isolates from Bangladesh and Vietnam were collected by collaborators in those countries. Genotyping. The PulseNet pulsed-field gel electrophoresis (PFGE) protocol for Salmonella and other enterobacteria was used for PFGE analysis of S. Typhi isolates (21). A smaller amount of XbaI (10 U) was used for
Received 14 June 2014 Returned for modification 11 July 2014 Accepted 10 August 2014 Published ahead of print 18 August 2014 Address correspondence to Chien-Shun Chiou, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.03608-14
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We characterized Salmonella enterica serovar Typhi isolates from Bangladesh, Indonesia, Taiwan, and Vietnam to investigate their genetic relatedness and antimicrobial resistance. The isolates from Bangladesh and Vietnam were genetically closely related but were distant from those from Indonesia and Taiwan. All but a few isolates from Indonesia and Taiwan were susceptible to all antimicrobials tested. The majority of isolates from Bangladesh and Vietnam were multidrug resistant (MDR) and belonged to the widespread haplotype H58 clone. IncHI1 plasmids were detected in all MDR S. Typhi isolates from Vietnam but in only 15% of MDR isolates from Bangladesh. Resistance genes in the majority of MDR S. Typhi isolates from Bangladesh should reside in the chromosome. Among the isolates from Bangladesh, 82% and 40% were resistant to various concentrations of nalidixic acid and ciprofloxacin, respectively. Several resistance mechanisms, including alterations in gyrase A, the presence of QnrS, and enhanced efflux pumps, were involved in the reduced susceptibility and resistance to fluoroquinolones. Intensive surveillance is necessary to monitor the spread of chromosome-mediated MDR and fluoroquinolone-resistant S. Typhi emerging in Bangladesh.
Chiou et al.
RESULTS
Antimicrobial resistance. All 180 isolates were susceptible to aztreonam, cefotaxime, ceftazidime, imipenem, and kanamycin (Table 1). Only 1 isolate was resistant to gentamicin. The resistance rates for the remaining 8 antimicrobials tested ranged from
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TABLE 1 Antimicrobial resistance in S. Typhi isolates from four Asian countries % of isolates with resistance Antimicrobial
Bangladesh Indonesia Taiwan Vietnam Total (n ⫽ 38) (n ⫽ 55) (n ⫽ 36) (n ⫽ 51) (n ⫽ 180)
Aztreonam Cefotaxime Ceftazidime Imipenem Nalidixic acid Ciprofloxacin Gentamicin Kanamycin Ampicillin Chloramphenicol Streptomycin Sulfamethoxazole Tetracycline Trimethoprim
0.0 0.0 0.0 0.0 81.6 39.5 0.0 0.0 68.4 57.9 60.5 68.4 21.1 57.9
0.0 0.0 0.0 0.0 1.8 0.0 1.8 0.0 1.8 3.6 3.6 3.6 3.6 1.8
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8 0.0 0.0 2.8 2.8 2.8
0.0 0.0 0.0 0.0 19.6 0.0 0.0 0.0 80.4 80.4 80.4 80.4 84.3 80.4
0.0 0.0 0.0 0.0 23.3 8.3 0.6 0.0 38.3 36.1 36.7 38.9 30.0 36.1
8.3% for ciprofloxacin to 38.9% for sulfamethoxazole. The resistance rates were significantly different among originating countries. Nalidixic acid resistance was observed in 81.6% of isolates from Bangladesh and in 19.6% of isolates from Vietnam. Ciprofloxacin resistance was observed in only 15 (39.5%) isolates from Bangladesh. The majority of isolates from Bangladesh and Vietnam displayed resistance to the pentadrugs ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT) and to trimethoprim. The resistance rates of these 6 antimicrobials were 80.4% to 84.3% for isolates from Vietnam and 21.1% to 68.4% for isolates from Bangladesh. Clonal relationships among isolates. The genetic relatedness among the S. Typhi isolates was assessed based on PFGE patterns and MLVA5 profiles resulting from an analysis of 5 less-variable VNTRs (Sty2, Sty3, Sty20, Sty39, and Sty42) (22). The dendrogram in Fig. 1 was constructed using the composite of PFGE patterns and MLVA5 profiles in a 1:1 weight ratio. Four distinct clusters, A, B, C, and D, were formed from 171 of the 180 isolates. The clusters were correlated with countries of origin and distinct levels of antimicrobial resistance. Isolates in cluster A were primarily from Taiwan and were susceptible to all antimicrobials tested or resistant to only one single agent (Fig. 1). Isolates in cluster B comprised isolates from Bangladesh and Vietnam, and 85% (67/79 isolates) of the isolates were MDR. Twenty-nine (74%) of the isolates from Bangladesh and 49 (96%) from Vietnam were located in cluster B. Cluster C consisted of 15 isolates from Taiwan, 4 from Bangladesh, 4 from Indonesia, and only 1 from Vietnam. The isolates from Taiwan and Indonesia were pan-susceptible, while those from Bangladesh and Vietnam were either MDR or resistant to nalidixic acid. The isolates in cluster D were primarily from Indonesia and Taiwan, and the majority were susceptible to all antimicrobials tested. Phylogenetic analysis. The phylogenetic relationships among the strains were established using 1,787 SNPs and UPGMA. As shown in Fig. 2, strains Bd1380 and Bd1980 were most closely related to strains with haplotype H58. Resistance patterns and resistance genes. A total of 15 antibiogram types were discovered for the 180 S. Typhi isolates (Table 2). Twelve types (A1 to A6 and A9 to A15), comprising 71 isolates,
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digestion of each sample. PFGE images were recorded digitally in tiff file format, using a Gel Logic 212 Pro imaging system (Carestream Health Inc., Rochester, NY). A previously described multilocus variable-number tandem-repeat analysis (MLVA) protocol (22) was used to characterize the S. Typhi isolates. To investigate the phylogenetic relationships among the 19 S. Typhi strains described by Holt et al. (23) and the prevalent MDR strains from Bangladesh and Vietnam, two MDR strains from Bangladesh, Bd1380 (antibiogram type A11) and Bd1980 (type A2), were subjected to whole-genome sequencing using the Ion Torrent platform, which was performed by a local biotechnology company. The sequence data from the reads provided over 200⫻ coverage of the S. Typhi genome. Genotyping data analysis. PFGE images were analyzed using the fingerprint analysis software BioNumerics, version 6.6 (Applied Maths, Kortrijk, Belgium). The number of repeats for each allele of the variablenumber tandem repeats (VNTRs) was converted from the amplicon size and saved as “Character Type” in the BioNumerics database. A dendrogram was constructed using composite data sets of PFGE-XbaI patterns and MLVA5 profiles in a 1:1 weight ratio. MLVA5 was based on a panel of five slowly evolving VNTRs: Sty2, Sty3, Sty20, Sty39, and Sty42. The 1,787 single-nucleotide polymorphisms (SNPs) used for phylogenetic analysis were identified from the sequence reads for strains Bd1380 and Bd1980, according to the method described in the study of Holt et al. (23). The phylogenetic relationships among strains were constructed using 1,787 SNPs and the unweighted-pair group method using average linkages (UPGMA). AST. S. Typhi isolates were subjected to antimicrobial susceptibility testing (AST) with 14 antimicrobials, using the broth microdilution method and custom-made 96-well Sensititre MIC panels (Trek Diagnostic Systems Ltd., West Grinstead, England). The test procedure was performed according to the manufacturer’s instructions. The 14 antimicrobials included ampicillin, aztreonam, cefotaxime, ceftazidime, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, imipenem, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim. The 2013 Clinical and Laboratory Standards Institute (24) interpretive criteria were used, except in the case of streptomycin, for which MICs of ⭌64, 32, and ⬉16 g/ml were defined as thresholds for resistant, intermediate, and susceptible results, respectively. Detection of resistance genes and the IncHI1 plasmid. Previously described PCR protocols were applied to detect the resistance genes blaTEM-1, catA1, strA-strB, sul1, tetA(B), dhfrA7 (25), sul2, and tetA (26) and the IncHI1 plasmid (27). The method of Kado and Liu (28) was used to analyze plasmid profiles of MDR S. Typhi isolates. Southern hybridization was performed on the MDR S. Typhi isolates from Bangladesh, following the protocol of a DIG High Prime DNA labeling and detection starter kit (Roche). Characterization of nalidixic acid and ciprofloxacin resistance mechanisms. To characterize the mechanisms of nalidixic acid and ciprofloxacin resistance in the 38 S. Typhi isolates from Bangladesh, mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA, gyrB, parC, and parE genes and the presence of qnrA, qnrB1, qnrB4, qnrD, and qnrS were analyzed, and the effect of the efflux pump on nalidixic acid and ciprofloxacin resistance was assessed using the efflux pump inhibitor phenylalanine-arginine -naphthylamide (PaN). The primers described by Kim et al. (29) were used for PCR amplification and DNA sequencing of the gyrase and topoisomerase IV genes and for PCR detection of the qnr genes. The effect of the efflux pump on resistance was determined by comparing MICs of nalidixic acid and ciprofloxacin in either the presence or absence of 25 g/ml PaN, using Sensititre MIC panels (30).
Antimicrobial Resistance in S. Typhi
Downloaded from http://aac.asm.org/ on November 6, 2014 by Stellenbosch University FIG 1 Genetic relationships among 180 S. Typhi isolates from four countries, with corresponding antimicrobial susceptibility patterns. The dendrogram was constructed based on the PFGE patterns and MLVA5 profiles in a 1:1 weight ratio, using the UPGMA algorithm provided in BioNumerics software, with settings of 1% optimization and 0.95% tolerance. MLVA5 was based on a panel of five slowly-evolving VNTRs (Sty2, Sty3, Sty20, Sty39, and Sty42). With respect to antimicrobial susceptibility, resistance is denoted by red rectangles, intermediate resistance by yellow rectangles, and susceptibility by green rectangles. The country of origin for each isolate is marked in cyan for Bangladesh, deep blue for Indonesia, white for Taiwan, and pink for Vietnam. Abbreviations: AMP, ampicillin; ATM, aztreonam; FTX, cefotaxime; CAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; KAN, kanamycin; IMP, imipenem; NAL, nalidixic acid; STR, streptomycin; SUL, sulfamethoxazole; TCY, tetracycline; TMP, trimethoprim.
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⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ 35 53
1 11 1 3 2 6 8 2 1
4
1 1
1 11 1 3 9 6 11 35 1 1 1 1 1 1 97 GEN-NAL-AMP-CHL-STR-SUL-TCY-TMP CIP-NAL-AMP-CHL-STR-SUL-TMP CIP-NAL-AMP-STR-SUL-TCY CIP-NAL-AMP-SUL-TCY NAL-AMP-CHL-STR-SUL-TCY-TMP NAL-AMP-CHL-STR-SUL-TMP NAL AMP-CHL-STR-SUL-TCY-TMP AMP-CHL-STR-SUL-TMP AMP-STR-SUL-TCY AMP-SUL-TCY-TMP CHL-STR-SUL-TCY CHL-TCY-TMP TCY Pan-susceptible A1 A2 A3 A4 A5 A6 A8 A9 A11 A12 A13 A14 A15 A19 A20
Abbreviations: AMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; NAL, nalidixic acid; STR, streptomycin; SUL, sulfamethoxazole; TCY, tetracycline; TMP, trimethoprim. b ⫹, present; ⫺, absent; (⫺), resistance not contributed by the indicated gene.
1
7
1 1 5
(⫺) ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫺ (⫺) ⫹ ⫺ ⫺ 3 33
catA1 blaTEM-1 Vietnam (n ⫽ 51) Taiwan (n ⫽ 36) Indonesia (n ⫽ 55)
No. of isolates from:
Bangladesh (n ⫽ 38) No. of isolates Resistance patterna Antibiogram type
a
dfrA7
(⫺) ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺
tetA(B) tetA
⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺
sul2 aac.asm.org
strAB
sul1 6504
TABLE 2 Distribution of resistance patterns and presence of resistance genes in S. Typhi isolates
were MDR, and these types were detected in 68.4% (26/38 isolates) of isolates from Bangladesh and 80.4% (42/51 isolates) of isolates from Vietnam. Of the antibiogram types with MDR patterns, A5 and A9 were shared by isolates across Bangladesh and Vietnam. The 15 ciprofloxacin-resistant isolates fell into 3 antibiogram types: A2, A3, and A4. Compared with isolates from Bangladesh and Vietnam, those from Indonesia and Taiwan displayed low resistance rates to the antimicrobials tested. Only three isolates from the two countries were MDR. The genes blaTEM-1, catA1, strA-strB, sul1 and sul2, tetA and tetA(B), and dfrA7 contributed primarily to resistances of the S. Typhi isolates to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim, respectively (Table 2). Exceptions were that chloramphenicol resistance in the isolates with antibiograms A1 and A14 was not mediated by catA1, streptomycin resistance in isolates with types A3 and A14 was not attributed to strA-strB, and trimethoprim resistance in isolates with type A1 was not caused by dfrA7. IncHI1 plasmids were detected in 15.4% (4/26 isolates) of MDR isolates from Bangladesh and 100% (42/42 isolates) of MDR isolates from Vietnam. The isolates with types A5 and A9 shared the same set of resistance genes [blaTEM-1, catA1, strA-strB, sul1, tetA(B), and dfrA7] for ACSSuT and trimethoprim. The isolates with types A2, A6, and A11 did not harbor any IncHI1 plasmid but shared the same set of resistance genes (blaTEM-1, catA1, strA-strB, sul1, sul2, and dfrA7) for resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and trimethoprim. Using the method of Kado and Liu for plasmid profile analysis, no plasmids were detected in the MDR S. Typhi isolates having antibiogram types A2 and A11. Southern hybridization analysis using a PCR-amplified DNA fragment of blaTEM-1 as a probe indicated that the resistance gene was located on plasmids for the MDR S. Typhi isolates from Bangladesh with antibiogram types A3, A4, A5, and A9 but was located on the chromosome for those with types A2, A6, and A11. Characterization of nalidixic acid and ciprofloxacin resistance mechanisms in isolates from Bangladesh. The 38 isolates from Bangladesh were categorized into the following 5 ciprofloxacin susceptibility groups: a susceptible group, with MICs of ⱕ0.03
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺
Presence of gene or plasmidb
structed based on 1,787 SNPs, using the UPGMA algorithm.
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FIG 2 Genetic relatedness among S. Typhi strains. The dendrogram was con-
⫹ ⫹ (⫺) ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ (⫺) ⫺ ⫺ ⫺
IncHI1
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Antimicrobial Resistance in S. Typhi
TABLE 3 Characteristics of resistance to nalidixic acid and ciprofloxacin in 38 S. Typhi isolates from Bangladesh Ciprofloxacin MIC/ nalidixic acid MIC (g/ml)
No. of isolates
qnrS
gyrA
parC
Ciprofloxacin
Nalidixic acid
⬉0.03/⬉4 0.12–0.5/128–256 4/256 or ⬎256 8/⬎256 16/⬎256
7 16 4 2 9
⫺ ⫺ ⫹ ⫺ ⫺
⫺ S83F, S83Y, or D87N S83Y S83F ⫹ D87G S83F ⫹ D87G
⫺ ⫺ ⫺ E92K E92K
1 1 2–4 1 2
1 2–8 4 4 4–8
a
Presence of gene or encoded mutationsa
Fold MIC reduction with PaN
⫹, present; ⫺, absent.
DISCUSSION
We compared the genotypes and antimicrobial susceptibility patterns of S. Typhi isolates from four Asian countries (Bangladesh, Indonesia, Taiwan, and Vietnam) and revealed that the majority of isolates from Bangladesh and Vietnam are genetically closely related but are distant from those from Indonesia and Taiwan. The majority of isolates from Bangladesh and Vietnam are MDR and have developed resistance to nalidixic acid (Table 1). All MDR strains from these two countries fall into a common genetic group (Fig. 1). SNP analysis indicates that these MDR S. Typhi strains should belong to haplotype H58 (23). The H58 clone typically shows reduced susceptibility to fluoroquinolones and has spread widely over the Indochina Peninsula, the Indian subcontinent, and Africa (18–20, 23, 31) but is not prevalent in Indonesia (32). The MDR S. Typhi isolates from Bangladesh and Vietnam were resistant primarily to ACSSuT and trimethoprim (Fig. 1). The resistances to the 6 drugs were associated with the resistance genes blaTEM-1, catA1, strA-strB, sul1, sul2, tetA, tetA(B), and dfrA7, which are frequently carried on IncHI1 plasmids (13). This plasmid type is frequently associated with globally spread MDR S. Typhi and MDR S. Paratyphi A (15, 16). In this study, all MDR isolates and a tetracycline-resistant isolate from Vietnam carried IncHI1 plasmids, whereas only 4 of the 26 MDR isolates from Bangladesh carried an IncHI1-type plasmid (Table 2). IncHI1 plasmids were not detected in the isolates with MDR patterns A2, A3, A4, A6, and A11, which were identified in the isolates from Bangladesh. The resistance genes may have been carried on other incompatibility plasmids (14). However, the isolates with types A2 and A11 were not observed to be associated with any plasmid, as
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analyzed using the method of Kado and Liu (28), indicating that the resistance genes were chromosomal. Southern hybridization analysis using blaTEM-1 DNA as a probe supported the plasmid analysis findings and revealed that the MDR genes in the type A2, A6, and A11 isolates were carried on the chromosome. Resistance to quinolones and fluoroquinolones in bacteria may result from alterations in target enzymes (DNA gyrase and topoisomerase IV), target protection by Qnr proteins, and/or decreased intracellular accumulation of antimicrobials mediated by enhanced efflux pump activity and a decrease in cell membrane permeability (33). A high level of resistance to the drugs may require multiple amino acid changes in gyrase and topoisomerase IV, in addition to combinations of other mechanisms, including changes in active efflux pumps and porins and acquisition of Qnr proteins (10, 34, 35). In this study, the S. Typhi isolates from Bangladesh displayed resistance to various concentrations of nalidixic acid and ciprofloxacin, mediated by combinations of several resistance mechanisms (Table 3). Resistance to nalidixic acid was associated with alterations in gyrase and an enhanced efflux pump activity for nalidixic acid. Mutations in gyrase A may also result in a reduced susceptibility to ciprofloxacin (MIC ⫽ 0.125, 0.25, or 0.5 g/ml). The resistance to ciprofloxacin at 4 g/ml in 4 isolates was associated with a single mutation in gyrase A, the presence of the QnrS protein, and an enhanced efflux pump activity for ciprofloxacin. Two amino acid substitutions in gyrase A and a substitution at topoisomerase IV are likely required for resistance to ciprofloxacin at 8 g/ml, and an enhanced efflux pump activity is additionally required for resistance to ciprofloxacin at 16 g/ml. With respect to fluoroquinolone resistance, mutations in parC typically occur at codons 57, 66, and 80 (36); the role of the mutation at codon 92 (encoding an E92K mutation) in fluoroquinolone resistance requires further investigation. These complicated mechanisms suggest that the quinolone- and fluoroquinolone-resistant strains in Bangladesh emerged independently of one another, although they are genetically closely related. Typhoid fever is rare in Taiwan, a country with a population of 23 million. From 2000 to 2013, 19 to 80 cases were confirmed each year, and at least 39% of these cases were imported. The majority of cases were from Indonesia; for example, 60 of the 80 cases in 2009 were in Indonesian migrant workers (37). In this study, the majority of Indonesian isolates were recovered from Indonesian migrant workers in Taiwan. S. Typhi isolates were obtained in routine health examinations within 3 days after the workers entered Taiwan, and some isolates were recovered from typhoid carriers due to epidemiological investigation of typhoid cases. MDR S. Typhi and ciprofloxacin-resistant S. Typhi strains were noted in Surabaya, Indonesia, in 2008 (38). However, 53 of the 55 Indonesian isolates characterized in this study were pan-susceptible
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g/ml; a group with reduced susceptibility, with MICs of 0.12 to 0.5 g/ml; and resistant groups, with MICs of 4 g/ml, 8 g/ml, and 16 g/ml (Table 3). The isolates with reduced susceptibility to ciprofloxacin were resistant to nalidixic acid (MICs of 128 to 256 g/ml), and the trait of resistance was associated with a mutation in gyrase subunit A (S83F, S83Y, or D87N) and enhanced activity of the efflux pump for nalidixic acid. The isolates with a ciprofloxacin MIC of 4 g/ml were highly resistant to nalidixic acid (MICs of ⱖ256 g/ml), and the resistance was associated with a mutation (S83Y) in gyrase A, the presence of qnrS, and enhanced activity of the efflux pumps for ciprofloxacin and nalidixic acid. The isolates with ciprofloxacin MICs of 8 and 16 g/ml also displayed high nalidixic acid resistance (MICs of ⬎256 g/ml), carried two mutations (S83F and D87G) in gyrase A and one mutation (E92K) in topoisomerase IV, and showed enhanced efflux pump activity for nalidixic acid. The isolates with a ciprofloxacin MIC of 16 g/ml also showed enhanced efflux pump activity for ciprofloxacin.
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ACKNOWLEDGMENTS This work was supported by a grant (DOH98-DC-2021) from the Centers for Disease Control, Department of Health, Taiwan, a grant (MOHW103CDC-C-315-000801) from the Ministry of Health and Welfare, Taiwan, an intramural grant (IDPP03-014) from the National Health Research Institutes, and a grant (H17-Shinkou-Ippan-019) from the Ministry of Health, Labor and Welfare, Japan. We sincerely thank The International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), for kindly providing S. Typhi isolates for this study. We declare that there are no conflicts of interest regarding the publication of this paper.
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7. Smith AM, Govender N, Keddy KH. 2010. Quinolone-resistant Salmonella Typhi in South Africa, 2003–2007. Epidemiol. Infect. 138:86 –90. http://dx.doi.org/10.1017/S0950268809990331. 8. Menezes GA, Harish BN, Khan MA, Goessens WH, Hays JP. 2012. Antimicrobial resistance trends in blood culture positive Salmonella Typhi isolates from Pondicherry, India, 2005–2009. Clin. Microbiol. Infect. 18:239 –245. http://dx.doi.org/10.1111/j.1469-0691.2011.03546.x. 9. Saha SK, Darmstadt GL, Baqui AH, Crook DW, Islam MN, Islam M, Hossain M, El Arifeen S, Santosham M, Black RE. 2006. Molecular basis of resistance displayed by highly ciprofloxacin-resistant Salmonella enterica serovar Typhi in Bangladesh. J. Clin. Microbiol. 44:3811–3813. http: //dx.doi.org/10.1128/JCM.01197-06. 10. Keddy KH, Smith AM, Sooka A, Ismail H, Oliver S. 2010. Fluoroquinolone-resistant typhoid, South Africa. Emerg. Infect. Dis. 16:879 – 880. http://dx.doi.org/10.3201/eid1605.091917. 11. Lee CJ, Su LH, Huang YC, Chiu CH. 2013. First isolation of ciprofloxacinresistant Salmonella enterica serovar Typhi in Taiwan. J. Microbiol. Immunol. Infect. 46:469 – 473. http://dx.doi.org/10.1016/j.jmii.2013.01.002. 12. Medalla F, Sjolund-Karlsson M, Shin S, Harvey E, Joyce K, Theobald L, Nygren BN, Pecic G, Gay K, Austin J, Stuart A, Blanton E, Mintz ED, Whichard JM, Barzilay EJ. 2011. Ciprofloxacin-resistant Salmonella enterica serotype Typhi, United States, 1999 –2008. Emerg. Infect. Dis. 17: 1095–1098. http://dx.doi.org/10.3201/eid/1706.100594. 13. Wain J, Diem Nga LT, Kidgell C, James K, Fortune S, Song Diep T, Ali T, O Gaora P, Parry C, Parkhill J, Farrar J, White NJ, Dougan G. 2003. Molecular analysis of incHI1 antimicrobial resistance plasmids from Salmonella serovar Typhi strains associated with typhoid fever. Antimicrob. Agents Chemother. 47:2732–2739. http://dx.doi.org/10.1128/AAC.47.9 .2732-2739.2003. 14. Mirza S, Kariuki S, Mamun KZ, Beeching NJ, Hart CA. 2000. Analysis of plasmid and chromosomal DNA of multidrug-resistant Salmonella enterica serovar Typhi from Asia. J. Clin. Microbiol. 38:1449 –1452. 15. Holt KE, Thomson NR, Wain J, Phan MD, Nair S, Hasan R, Bhutta ZA, Quail MA, Norbertczak H, Walker D, Dougan G, Parkhill J. 2007. Multidrug-resistant Salmonella enterica serovar Paratyphi A harbors IncHI1 plasmids similar to those found in serovar Typhi. J. Bacteriol. 189: 4257– 4264. http://dx.doi.org/10.1128/JB.00232-07. 16. Holt KE, Phan MD, Baker S, Duy PT, Nga TV, Nair S, Turner AK, Walsh C, Fanning S, Farrell-Ward S, Dutta S, Kariuki S, Weill FX, Parkhill J, Dougan G, Wain J. 2011. Emergence of a globally dominant IncHI1 plasmid type associated with multiple drug resistant typhoid. PLoS Negl. Trop. Dis. 5:e1245. http://dx.doi.org/10.1371/journal.pntd .0001245. 17. Le TA, Fabre L, Roumagnac P, Grimont PA, Scavizzi MR, Weill FX. 2007. Clonal expansion and microevolution of quinolone-resistant Salmonella enterica serotype Typhi in Vietnam from 1996 to 2004. J. Clin. Microbiol. 45:3485–3492. http://dx.doi.org/10.1128/JCM.00948-07. 18. Holt KE, Baker S, Dongol S, Basnyat B, Adhikari N, Thorson S, Pulickal AS, Song Y, Parkhill J, Farrar JJ, Murdoch DR, Kelly DF, Pollard AJ, Dougan G. 2010. High-throughput bacterial SNP typing identifies distinct clusters of Salmonella Typhi causing typhoid in Nepalese children. BMC Infect. Dis. 10:144. http://dx.doi.org/10.1186/1471-2334-10-144. 19. Roumagnac P, Weill FX, Dolecek C, Baker S, Brisse S, Chinh NT, Le TA, Acosta CJ, Farrar J, Dougan G, Achtman M. 2006. Evolutionary history of Salmonella Typhi. Science 314:1301–1304. http://dx.doi.org/10 .1126/science.1134933. 20. Kariuki S, Revathi G, Kiiru J, Mengo DM, Mwituria J, Muyodi J, Munyalo A, Teo YY, Holt KE, Kingsley RA, Dougan G. 2010. Typhoid in Kenya is associated with a dominant multidrug-resistant Salmonella enterica serovar Typhi haplotype that is also widespread in Southeast Asia. J. Clin. Microbiol. 48:2171–2176. http://dx.doi.org/10.1128/JCM.01983-09. 21. Ribot EM, Fair MA, Gautom R, Cameron DN, Hunter SB, Swaminathan B, Barrett TJ. 2006. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog. Dis. 3:59 – 67. http://dx.doi .org/10.1089/fpd.2006.3.59. 22. Tien YY, Ushijima H, Mizuguchi M, Liang SY, Chiou CS. 2012. Use of multilocus variable-number tandem repeat analysis in molecular subtyping of Salmonella enterica serovar Typhi isolates. J. Med. Microbiol. 61: 223–232. http://dx.doi.org/10.1099/jmm.0.037291-0. 23. Holt KE, Parkhill J, Mazzoni CJ, Roumagnac P, Weill FX, Goodhead I, Rance R, Baker S, Maskell DJ, Wain J, Dolecek C, Achtman M, Dougan G. 2008. High-throughput sequencing provides insights into genome
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(Table 2), and the majority were very clonal (Fig. 1), suggesting that a typhoid fever outbreak may have occurred at that time in the area from which the immigrant workers originated. Recently, typhoid fever cases in Taiwan have more often been linked to longterm S. Typhi carriers from Indonesia (39). In this study, 5 genotypes were found in isolates from Indonesian workers and Taiwanese typhoid patients. The genotyping data suggest that the introduction of Indonesian workers into Taiwan has affected S. Typhi infections in Taiwan. Conclusions. Almost all S. Typhi isolates from Taiwan and Indonesia are susceptible to all antimicrobials tested. Some isolates from the two countries share identical genotypic profiles, suggesting a close epidemiological relationship between migrant workers and typhoid patients in Taiwan. In contrast, the majority of S. Typhi isolates from Bangladesh and Vietnam are MDR and belong to haplotype H58, a prevalent MDR clone that has spread over all of Asia and Africa. In more than 50% of these MDR isolates from Bangladesh, the genes responsible for resistance reside on the chromosome. Among the S. Typhi isolates from Bangladesh, 82% and 40% were resistant to nalidixic acid and ciprofloxacin, respectively. Intensive surveillance is particularly required to monitor the spread of chromosome-mediated MDR and fluoroquinolone-resistant S. Typhi that has emerged in Bangladesh.
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24.
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32. Baker S, Holt K, van de Vosse E, Roumagnac P, Whitehead S, King E, Ewels P, Keniry A, Weill FX, Lightfoot D, van Dissel JT, Sanderson KE, Farrar J, Achtman M, Deloukas P, Dougan G. 2008. High-throughput genotyping of Salmonella enterica serovar Typhi allowing geographical assignment of haplotypes and pathotypes within an urban district of Jakarta, Indonesia. J. Clin. Microbiol. 46:1741–1746. http://dx.doi.org/10 .1128/JCM.02249-07. 33. Jacoby GA. 2005. Mechanisms of resistance to quinolones. Clin. Infect. Dis. 41(Suppl 2):S120 –S126. http://dx.doi.org/10.1086/428052. 34. Kanematsu E, Deguchi T, Yasuda M, Kawamura T, Nishino Y, Kawada Y. 1998. Alterations in the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV associated with quinolone resistance in Enterococcus faecalis. Antimicrob. Agents Chemother. 42:433. 35. Tran JH, Jacoby GA. 2002. Mechanism of plasmid-mediated quinolone resistance. Proc. Natl. Acad. Sci. U. S. A. 99:5638 –5642. http://dx.doi.org /10.1073/pnas.082092899. 36. Eaves DJ, Randall L, Gray DT, Buckley A, Woodward MJ, White AP, Piddock LJ. 2004. Prevalence of mutations within the quinolone resistance-determining region of gyrA, gyrB, parC, and parE and association with antibiotic resistance in quinolone-resistant Salmonella enterica. Antimicrob. Agents Chemother. 48:4012– 4015. http://dx.doi.org/10.1128 /AAC.48.10.4012-4015.2004. 37. Wu L-J, Chen W-C, Lin M-C, Yang S-Y. 2011. The epidemic and interventions in imported typhoid among Indonesian labors in 2009. Taiwan Epidemiol. Bull. 27:67–74. 38. Yanagi D, de Vries GC, Rahardjo D, Alimsardjono L, Wasito EB, De I, Kinoshita S, Hayashi Y, Hotta H, Osawa R, Kawabata M, Shirakawa T. 2009. Emergence of fluoroquinolone-resistant strains of Salmonella enterica in Surabaya, Indonesia. Diagn. Microbiol. Infect. Dis. 64:422– 426. http://dx.doi.org/10.1016/j.diagmicrobio.2009.04.006. 39. Chiou CS, Wei HL, Mu JJ, Liao YS, Liang SY, Liao CH, Tsao CS, Wang SC. 2013. Salmonella enterica serovar Typhi variants in long-term carriers. J. Clin. Microbiol. 51:669 – 672. http://dx.doi.org/10.1128/JCM.02726-12.
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variation and evolution in Salmonella Typhi. Nat. Genet. 40:987–993. http://dx.doi.org/10.1038/ng.195. Clinical and Laboratory Standards Institute (CLSI). 2013. Performance standards for antimicrobial susceptibility testing; 23rd informational supplement. CLSI document M100-S23. Clinical and Laboratory Standards Institute, Wayne, PA. Tamang MD, Oh JY, Seol SY, Kang HY, Lee JC, Lee YC, Cho DT, Kim J. 2007. Emergence of multidrug-resistant Salmonella enterica serovar Typhi associated with a class 1 integron carrying the dfrA7 gene cassette in Nepal. Int. J. Antimicrob. Agents 30:330 –335. http://dx.doi.org/10.1016/j .ijantimicag.2007.05.009. Benacer D, Thong KL, Watanabe H, Puthucheary SD. 2010. Characterization of drug resistant Salmonella enterica serotype Typhimurium by antibiograms, plasmids, integrons, resistance genes and PFGE. J. Microbiol. Biotechnol. 20:1042–1052. http://dx.doi.org/10.4014/jmb.0910.10028. Kariuki S, Revathi G, Muyodi J, Mwituria J, Munyalo A, Mirza S, Hart CA. 2004. Characterization of multidrug-resistant typhoid outbreaks in Kenya. J. Clin. Microbiol. 42:1477–1482. http://dx.doi.org/10.1128/JCM .42.4.1477-1482.2004. Kado CI, Liu ST. 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145:1365–1373. Kim KY, Park JH, Kwak HS, Woo GJ. 2011. Characterization of the quinolone resistance mechanism in foodborne Salmonella isolates with high nalidixic acid resistance. Int. J. Food Microbiol. 146:52–56. http://dx .doi.org/10.1016/j.ijfoodmicro.2011.01.037. Lamers RP, Cavallari JF, Burrows LL. 2013. The efflux inhibitor phenylalanine-arginine beta-naphthylamide (PAbetaN) permeabilizes the outer membrane of gram-negative bacteria. PLoS One 8:e60666. http://dx.doi .org/10.1371/journal.pone.0060666. Holt KE, Dolecek C, Chau TT, Duy PT, La TT, Hoang NV, Nga TV, Campbell JI, Manh BH, Vinh Chau NV, Hien TT, Farrar J, Dougan G, Baker S. 2011. Temporal fluctuation of multidrug resistant Salmonella Typhi haplotypes in the Mekong River delta region of Vietnam. PLoS Negl. Trop. Dis. 5:e929. http://dx.doi.org/10.1371/journal.pntd.0000929.
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Chien-Shun Chiou, Tsai-Ling Lauderdale, Dac Cam Phung, Haruo Watanabe, Jung-Che Kuo, Pei-Jen Wang, Yen-Yi Liu, Shiu-Yun Liang and Pei-Chen Chen Antimicrob. Agents Chemother. 2014, 58(11):6501. DOI: 10.1128/AAC.03608-14. Published Ahead of Print 18 August 2014.
Antimicrobial Resistance in Salmonella enterica Serovar Typhi Isolates from Bangladesh, Indonesia, Taiwan, and Vietnam Chien-Shun Chiou,a Tsai-Ling Lauderdale,b Dac Cam Phung,c Haruo Watanabe,d Jung-Che Kuo,a Pei-Jen Wang,a Yen-Yi Liu,a Shiu-Yun Liang,a Pei-Chen Chenb Centers for Disease Control, Taichung, Taiwana; National Health Research Institutes, Zhunan, Taiwanb; National Institute of Hygiene and Epidemiology, Hanoi, Vietnamc; National Institute of Infectious Diseases, Tokyo, Japand
S
almonella enterica subspecies enterica serovar Typhi, the cause of typhoid fever, is transmitted primarily by ingestion of contaminated food and water. It was estimated to cause 21.6 million illnesses and 216,000 deaths during 2000 (1). Despite the fact that the incidence of typhoid fever has declined markedly in developed countries, it remains a major cause of morbidity in less-developed countries. South-central Asia and Southeast Asia are regions with high incidences of typhoid fever (⬎100/100,000 cases/year, respectively). In recent decades, cases of typhoid fever in developed countries have been associated predominantly with travelers returning from areas where typhoid fever is endemic (2, 3). Antimicrobials are the mainstay of therapy for typhoid patients. However, the intensive use of first-line antimicrobials, such as ampicillin, chloramphenicol, and cotrimoxazole, has led to the emergence and global spread of multidrug-resistant (MDR) S. Typhi strains (4). Due to increasing resistance to the antimicrobials used traditionally for therapy, the use of fluoroquinolones, such as ciprofloxacin, for the treatment of typhoid has become more common in developing countries. The use of fluoroquinolones has also led to a rapid increase in reduced susceptibility of S. Typhi to these therapeutics. MDR S. Typhi with reduced ciprofloxacin susceptibility has become common in Africa and South and Southeast Asia (3, 5–7). In recent years, ciprofloxacin-resistant S. Typhi has been reported frequently (8–12). MDR in S. Typhi is almost exclusively associated with selftransmissible IncHI1 plasmids, which carry a panel of genes conferring resistance to several first-line antimicrobials, including ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline, and trimethoprim (13, 14). This plasmid type has also been identified in MDR S. Paratyphi A (15). A study conducted by Holt et al. (16) indicated that prior to 1995, MDR typhoid was caused by a diverse range of S. Typhi and MDR plasmids. However, from 1995 onwards, 98% of MDR S. Typhi isolates were of S. Typhi haplotype H58, carrying the IncHI1 plasmid sequence type 6 (PST6). The PST6 plasmid confers on the host the ability to grow
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in high-salt medium, which may provide a competitive advantage over S. Typhi strains carrying other plasmid types. Currently, the majority of MDR S. Typhi H58 clone isolates are resistant to nalidixic acid, and they are widespread in Africa and Asia (17–20). In this study, we investigated the genetic relatedness and antimicrobial resistance among S. Typhi isolates from four Asian countries: Bangladesh, Indonesia, Taiwan, and Vietnam. The results indicate that the majority of S. Typhi isolates from Bangladesh and Vietnam are MDR and genetically closely related, 40% of isolates from Bangladesh are ciprofloxacin resistant, and several resistance mechanisms in the isolates from Bangladesh may underlie their resistance to nalidixic acid and ciprofloxacin. MATERIALS AND METHODS Bacterial isolates. Of 180 S. Typhi isolates included in this study, 38 were from Bangladesh and were collected in 2007, 55 were from Indonesia and were collected between 2007 and 2009, 36 were from Taiwan and were collected between 2007 and 2009, and 51 were from Vietnam and were collected between 2007 and 2008. The Indonesian isolates were collected in Taiwan by the Centers for Disease Control, Taiwan, from Indonesian migrant workers if the isolates were recovered within 60 days of their arrival to Taiwan and from returning travelers who visited only Indonesia and developed symptoms within 60 days. The isolates from Bangladesh and Vietnam were collected by collaborators in those countries. Genotyping. The PulseNet pulsed-field gel electrophoresis (PFGE) protocol for Salmonella and other enterobacteria was used for PFGE analysis of S. Typhi isolates (21). A smaller amount of XbaI (10 U) was used for
Received 14 June 2014 Returned for modification 11 July 2014 Accepted 10 August 2014 Published ahead of print 18 August 2014 Address correspondence to Chien-Shun Chiou, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.03608-14
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We characterized Salmonella enterica serovar Typhi isolates from Bangladesh, Indonesia, Taiwan, and Vietnam to investigate their genetic relatedness and antimicrobial resistance. The isolates from Bangladesh and Vietnam were genetically closely related but were distant from those from Indonesia and Taiwan. All but a few isolates from Indonesia and Taiwan were susceptible to all antimicrobials tested. The majority of isolates from Bangladesh and Vietnam were multidrug resistant (MDR) and belonged to the widespread haplotype H58 clone. IncHI1 plasmids were detected in all MDR S. Typhi isolates from Vietnam but in only 15% of MDR isolates from Bangladesh. Resistance genes in the majority of MDR S. Typhi isolates from Bangladesh should reside in the chromosome. Among the isolates from Bangladesh, 82% and 40% were resistant to various concentrations of nalidixic acid and ciprofloxacin, respectively. Several resistance mechanisms, including alterations in gyrase A, the presence of QnrS, and enhanced efflux pumps, were involved in the reduced susceptibility and resistance to fluoroquinolones. Intensive surveillance is necessary to monitor the spread of chromosome-mediated MDR and fluoroquinolone-resistant S. Typhi emerging in Bangladesh.
Chiou et al.
RESULTS
Antimicrobial resistance. All 180 isolates were susceptible to aztreonam, cefotaxime, ceftazidime, imipenem, and kanamycin (Table 1). Only 1 isolate was resistant to gentamicin. The resistance rates for the remaining 8 antimicrobials tested ranged from
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TABLE 1 Antimicrobial resistance in S. Typhi isolates from four Asian countries % of isolates with resistance Antimicrobial
Bangladesh Indonesia Taiwan Vietnam Total (n ⫽ 38) (n ⫽ 55) (n ⫽ 36) (n ⫽ 51) (n ⫽ 180)
Aztreonam Cefotaxime Ceftazidime Imipenem Nalidixic acid Ciprofloxacin Gentamicin Kanamycin Ampicillin Chloramphenicol Streptomycin Sulfamethoxazole Tetracycline Trimethoprim
0.0 0.0 0.0 0.0 81.6 39.5 0.0 0.0 68.4 57.9 60.5 68.4 21.1 57.9
0.0 0.0 0.0 0.0 1.8 0.0 1.8 0.0 1.8 3.6 3.6 3.6 3.6 1.8
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8 0.0 0.0 2.8 2.8 2.8
0.0 0.0 0.0 0.0 19.6 0.0 0.0 0.0 80.4 80.4 80.4 80.4 84.3 80.4
0.0 0.0 0.0 0.0 23.3 8.3 0.6 0.0 38.3 36.1 36.7 38.9 30.0 36.1
8.3% for ciprofloxacin to 38.9% for sulfamethoxazole. The resistance rates were significantly different among originating countries. Nalidixic acid resistance was observed in 81.6% of isolates from Bangladesh and in 19.6% of isolates from Vietnam. Ciprofloxacin resistance was observed in only 15 (39.5%) isolates from Bangladesh. The majority of isolates from Bangladesh and Vietnam displayed resistance to the pentadrugs ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT) and to trimethoprim. The resistance rates of these 6 antimicrobials were 80.4% to 84.3% for isolates from Vietnam and 21.1% to 68.4% for isolates from Bangladesh. Clonal relationships among isolates. The genetic relatedness among the S. Typhi isolates was assessed based on PFGE patterns and MLVA5 profiles resulting from an analysis of 5 less-variable VNTRs (Sty2, Sty3, Sty20, Sty39, and Sty42) (22). The dendrogram in Fig. 1 was constructed using the composite of PFGE patterns and MLVA5 profiles in a 1:1 weight ratio. Four distinct clusters, A, B, C, and D, were formed from 171 of the 180 isolates. The clusters were correlated with countries of origin and distinct levels of antimicrobial resistance. Isolates in cluster A were primarily from Taiwan and were susceptible to all antimicrobials tested or resistant to only one single agent (Fig. 1). Isolates in cluster B comprised isolates from Bangladesh and Vietnam, and 85% (67/79 isolates) of the isolates were MDR. Twenty-nine (74%) of the isolates from Bangladesh and 49 (96%) from Vietnam were located in cluster B. Cluster C consisted of 15 isolates from Taiwan, 4 from Bangladesh, 4 from Indonesia, and only 1 from Vietnam. The isolates from Taiwan and Indonesia were pan-susceptible, while those from Bangladesh and Vietnam were either MDR or resistant to nalidixic acid. The isolates in cluster D were primarily from Indonesia and Taiwan, and the majority were susceptible to all antimicrobials tested. Phylogenetic analysis. The phylogenetic relationships among the strains were established using 1,787 SNPs and UPGMA. As shown in Fig. 2, strains Bd1380 and Bd1980 were most closely related to strains with haplotype H58. Resistance patterns and resistance genes. A total of 15 antibiogram types were discovered for the 180 S. Typhi isolates (Table 2). Twelve types (A1 to A6 and A9 to A15), comprising 71 isolates,
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digestion of each sample. PFGE images were recorded digitally in tiff file format, using a Gel Logic 212 Pro imaging system (Carestream Health Inc., Rochester, NY). A previously described multilocus variable-number tandem-repeat analysis (MLVA) protocol (22) was used to characterize the S. Typhi isolates. To investigate the phylogenetic relationships among the 19 S. Typhi strains described by Holt et al. (23) and the prevalent MDR strains from Bangladesh and Vietnam, two MDR strains from Bangladesh, Bd1380 (antibiogram type A11) and Bd1980 (type A2), were subjected to whole-genome sequencing using the Ion Torrent platform, which was performed by a local biotechnology company. The sequence data from the reads provided over 200⫻ coverage of the S. Typhi genome. Genotyping data analysis. PFGE images were analyzed using the fingerprint analysis software BioNumerics, version 6.6 (Applied Maths, Kortrijk, Belgium). The number of repeats for each allele of the variablenumber tandem repeats (VNTRs) was converted from the amplicon size and saved as “Character Type” in the BioNumerics database. A dendrogram was constructed using composite data sets of PFGE-XbaI patterns and MLVA5 profiles in a 1:1 weight ratio. MLVA5 was based on a panel of five slowly evolving VNTRs: Sty2, Sty3, Sty20, Sty39, and Sty42. The 1,787 single-nucleotide polymorphisms (SNPs) used for phylogenetic analysis were identified from the sequence reads for strains Bd1380 and Bd1980, according to the method described in the study of Holt et al. (23). The phylogenetic relationships among strains were constructed using 1,787 SNPs and the unweighted-pair group method using average linkages (UPGMA). AST. S. Typhi isolates were subjected to antimicrobial susceptibility testing (AST) with 14 antimicrobials, using the broth microdilution method and custom-made 96-well Sensititre MIC panels (Trek Diagnostic Systems Ltd., West Grinstead, England). The test procedure was performed according to the manufacturer’s instructions. The 14 antimicrobials included ampicillin, aztreonam, cefotaxime, ceftazidime, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, imipenem, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim. The 2013 Clinical and Laboratory Standards Institute (24) interpretive criteria were used, except in the case of streptomycin, for which MICs of ⭌64, 32, and ⬉16 g/ml were defined as thresholds for resistant, intermediate, and susceptible results, respectively. Detection of resistance genes and the IncHI1 plasmid. Previously described PCR protocols were applied to detect the resistance genes blaTEM-1, catA1, strA-strB, sul1, tetA(B), dhfrA7 (25), sul2, and tetA (26) and the IncHI1 plasmid (27). The method of Kado and Liu (28) was used to analyze plasmid profiles of MDR S. Typhi isolates. Southern hybridization was performed on the MDR S. Typhi isolates from Bangladesh, following the protocol of a DIG High Prime DNA labeling and detection starter kit (Roche). Characterization of nalidixic acid and ciprofloxacin resistance mechanisms. To characterize the mechanisms of nalidixic acid and ciprofloxacin resistance in the 38 S. Typhi isolates from Bangladesh, mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA, gyrB, parC, and parE genes and the presence of qnrA, qnrB1, qnrB4, qnrD, and qnrS were analyzed, and the effect of the efflux pump on nalidixic acid and ciprofloxacin resistance was assessed using the efflux pump inhibitor phenylalanine-arginine -naphthylamide (PaN). The primers described by Kim et al. (29) were used for PCR amplification and DNA sequencing of the gyrase and topoisomerase IV genes and for PCR detection of the qnr genes. The effect of the efflux pump on resistance was determined by comparing MICs of nalidixic acid and ciprofloxacin in either the presence or absence of 25 g/ml PaN, using Sensititre MIC panels (30).
Antimicrobial Resistance in S. Typhi
Downloaded from http://aac.asm.org/ on November 6, 2014 by Stellenbosch University FIG 1 Genetic relationships among 180 S. Typhi isolates from four countries, with corresponding antimicrobial susceptibility patterns. The dendrogram was constructed based on the PFGE patterns and MLVA5 profiles in a 1:1 weight ratio, using the UPGMA algorithm provided in BioNumerics software, with settings of 1% optimization and 0.95% tolerance. MLVA5 was based on a panel of five slowly-evolving VNTRs (Sty2, Sty3, Sty20, Sty39, and Sty42). With respect to antimicrobial susceptibility, resistance is denoted by red rectangles, intermediate resistance by yellow rectangles, and susceptibility by green rectangles. The country of origin for each isolate is marked in cyan for Bangladesh, deep blue for Indonesia, white for Taiwan, and pink for Vietnam. Abbreviations: AMP, ampicillin; ATM, aztreonam; FTX, cefotaxime; CAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; KAN, kanamycin; IMP, imipenem; NAL, nalidixic acid; STR, streptomycin; SUL, sulfamethoxazole; TCY, tetracycline; TMP, trimethoprim.
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⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ 35 53
1 11 1 3 2 6 8 2 1
4
1 1
1 11 1 3 9 6 11 35 1 1 1 1 1 1 97 GEN-NAL-AMP-CHL-STR-SUL-TCY-TMP CIP-NAL-AMP-CHL-STR-SUL-TMP CIP-NAL-AMP-STR-SUL-TCY CIP-NAL-AMP-SUL-TCY NAL-AMP-CHL-STR-SUL-TCY-TMP NAL-AMP-CHL-STR-SUL-TMP NAL AMP-CHL-STR-SUL-TCY-TMP AMP-CHL-STR-SUL-TMP AMP-STR-SUL-TCY AMP-SUL-TCY-TMP CHL-STR-SUL-TCY CHL-TCY-TMP TCY Pan-susceptible A1 A2 A3 A4 A5 A6 A8 A9 A11 A12 A13 A14 A15 A19 A20
Abbreviations: AMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; NAL, nalidixic acid; STR, streptomycin; SUL, sulfamethoxazole; TCY, tetracycline; TMP, trimethoprim. b ⫹, present; ⫺, absent; (⫺), resistance not contributed by the indicated gene.
1
7
1 1 5
(⫺) ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫺ (⫺) ⫹ ⫺ ⫺ 3 33
catA1 blaTEM-1 Vietnam (n ⫽ 51) Taiwan (n ⫽ 36) Indonesia (n ⫽ 55)
No. of isolates from:
Bangladesh (n ⫽ 38) No. of isolates Resistance patterna Antibiogram type
a
dfrA7
(⫺) ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺
tetA(B) tetA
⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺
sul2 aac.asm.org
strAB
sul1 6504
TABLE 2 Distribution of resistance patterns and presence of resistance genes in S. Typhi isolates
were MDR, and these types were detected in 68.4% (26/38 isolates) of isolates from Bangladesh and 80.4% (42/51 isolates) of isolates from Vietnam. Of the antibiogram types with MDR patterns, A5 and A9 were shared by isolates across Bangladesh and Vietnam. The 15 ciprofloxacin-resistant isolates fell into 3 antibiogram types: A2, A3, and A4. Compared with isolates from Bangladesh and Vietnam, those from Indonesia and Taiwan displayed low resistance rates to the antimicrobials tested. Only three isolates from the two countries were MDR. The genes blaTEM-1, catA1, strA-strB, sul1 and sul2, tetA and tetA(B), and dfrA7 contributed primarily to resistances of the S. Typhi isolates to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim, respectively (Table 2). Exceptions were that chloramphenicol resistance in the isolates with antibiograms A1 and A14 was not mediated by catA1, streptomycin resistance in isolates with types A3 and A14 was not attributed to strA-strB, and trimethoprim resistance in isolates with type A1 was not caused by dfrA7. IncHI1 plasmids were detected in 15.4% (4/26 isolates) of MDR isolates from Bangladesh and 100% (42/42 isolates) of MDR isolates from Vietnam. The isolates with types A5 and A9 shared the same set of resistance genes [blaTEM-1, catA1, strA-strB, sul1, tetA(B), and dfrA7] for ACSSuT and trimethoprim. The isolates with types A2, A6, and A11 did not harbor any IncHI1 plasmid but shared the same set of resistance genes (blaTEM-1, catA1, strA-strB, sul1, sul2, and dfrA7) for resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and trimethoprim. Using the method of Kado and Liu for plasmid profile analysis, no plasmids were detected in the MDR S. Typhi isolates having antibiogram types A2 and A11. Southern hybridization analysis using a PCR-amplified DNA fragment of blaTEM-1 as a probe indicated that the resistance gene was located on plasmids for the MDR S. Typhi isolates from Bangladesh with antibiogram types A3, A4, A5, and A9 but was located on the chromosome for those with types A2, A6, and A11. Characterization of nalidixic acid and ciprofloxacin resistance mechanisms in isolates from Bangladesh. The 38 isolates from Bangladesh were categorized into the following 5 ciprofloxacin susceptibility groups: a susceptible group, with MICs of ⱕ0.03
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺
Presence of gene or plasmidb
structed based on 1,787 SNPs, using the UPGMA algorithm.
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FIG 2 Genetic relatedness among S. Typhi strains. The dendrogram was con-
⫹ ⫹ (⫺) ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ (⫺) ⫺ ⫺ ⫺
IncHI1
Chiou et al.
Antimicrobial Resistance in S. Typhi
TABLE 3 Characteristics of resistance to nalidixic acid and ciprofloxacin in 38 S. Typhi isolates from Bangladesh Ciprofloxacin MIC/ nalidixic acid MIC (g/ml)
No. of isolates
qnrS
gyrA
parC
Ciprofloxacin
Nalidixic acid
⬉0.03/⬉4 0.12–0.5/128–256 4/256 or ⬎256 8/⬎256 16/⬎256
7 16 4 2 9
⫺ ⫺ ⫹ ⫺ ⫺
⫺ S83F, S83Y, or D87N S83Y S83F ⫹ D87G S83F ⫹ D87G
⫺ ⫺ ⫺ E92K E92K
1 1 2–4 1 2
1 2–8 4 4 4–8
a
Presence of gene or encoded mutationsa
Fold MIC reduction with PaN
⫹, present; ⫺, absent.
DISCUSSION
We compared the genotypes and antimicrobial susceptibility patterns of S. Typhi isolates from four Asian countries (Bangladesh, Indonesia, Taiwan, and Vietnam) and revealed that the majority of isolates from Bangladesh and Vietnam are genetically closely related but are distant from those from Indonesia and Taiwan. The majority of isolates from Bangladesh and Vietnam are MDR and have developed resistance to nalidixic acid (Table 1). All MDR strains from these two countries fall into a common genetic group (Fig. 1). SNP analysis indicates that these MDR S. Typhi strains should belong to haplotype H58 (23). The H58 clone typically shows reduced susceptibility to fluoroquinolones and has spread widely over the Indochina Peninsula, the Indian subcontinent, and Africa (18–20, 23, 31) but is not prevalent in Indonesia (32). The MDR S. Typhi isolates from Bangladesh and Vietnam were resistant primarily to ACSSuT and trimethoprim (Fig. 1). The resistances to the 6 drugs were associated with the resistance genes blaTEM-1, catA1, strA-strB, sul1, sul2, tetA, tetA(B), and dfrA7, which are frequently carried on IncHI1 plasmids (13). This plasmid type is frequently associated with globally spread MDR S. Typhi and MDR S. Paratyphi A (15, 16). In this study, all MDR isolates and a tetracycline-resistant isolate from Vietnam carried IncHI1 plasmids, whereas only 4 of the 26 MDR isolates from Bangladesh carried an IncHI1-type plasmid (Table 2). IncHI1 plasmids were not detected in the isolates with MDR patterns A2, A3, A4, A6, and A11, which were identified in the isolates from Bangladesh. The resistance genes may have been carried on other incompatibility plasmids (14). However, the isolates with types A2 and A11 were not observed to be associated with any plasmid, as
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analyzed using the method of Kado and Liu (28), indicating that the resistance genes were chromosomal. Southern hybridization analysis using blaTEM-1 DNA as a probe supported the plasmid analysis findings and revealed that the MDR genes in the type A2, A6, and A11 isolates were carried on the chromosome. Resistance to quinolones and fluoroquinolones in bacteria may result from alterations in target enzymes (DNA gyrase and topoisomerase IV), target protection by Qnr proteins, and/or decreased intracellular accumulation of antimicrobials mediated by enhanced efflux pump activity and a decrease in cell membrane permeability (33). A high level of resistance to the drugs may require multiple amino acid changes in gyrase and topoisomerase IV, in addition to combinations of other mechanisms, including changes in active efflux pumps and porins and acquisition of Qnr proteins (10, 34, 35). In this study, the S. Typhi isolates from Bangladesh displayed resistance to various concentrations of nalidixic acid and ciprofloxacin, mediated by combinations of several resistance mechanisms (Table 3). Resistance to nalidixic acid was associated with alterations in gyrase and an enhanced efflux pump activity for nalidixic acid. Mutations in gyrase A may also result in a reduced susceptibility to ciprofloxacin (MIC ⫽ 0.125, 0.25, or 0.5 g/ml). The resistance to ciprofloxacin at 4 g/ml in 4 isolates was associated with a single mutation in gyrase A, the presence of the QnrS protein, and an enhanced efflux pump activity for ciprofloxacin. Two amino acid substitutions in gyrase A and a substitution at topoisomerase IV are likely required for resistance to ciprofloxacin at 8 g/ml, and an enhanced efflux pump activity is additionally required for resistance to ciprofloxacin at 16 g/ml. With respect to fluoroquinolone resistance, mutations in parC typically occur at codons 57, 66, and 80 (36); the role of the mutation at codon 92 (encoding an E92K mutation) in fluoroquinolone resistance requires further investigation. These complicated mechanisms suggest that the quinolone- and fluoroquinolone-resistant strains in Bangladesh emerged independently of one another, although they are genetically closely related. Typhoid fever is rare in Taiwan, a country with a population of 23 million. From 2000 to 2013, 19 to 80 cases were confirmed each year, and at least 39% of these cases were imported. The majority of cases were from Indonesia; for example, 60 of the 80 cases in 2009 were in Indonesian migrant workers (37). In this study, the majority of Indonesian isolates were recovered from Indonesian migrant workers in Taiwan. S. Typhi isolates were obtained in routine health examinations within 3 days after the workers entered Taiwan, and some isolates were recovered from typhoid carriers due to epidemiological investigation of typhoid cases. MDR S. Typhi and ciprofloxacin-resistant S. Typhi strains were noted in Surabaya, Indonesia, in 2008 (38). However, 53 of the 55 Indonesian isolates characterized in this study were pan-susceptible
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g/ml; a group with reduced susceptibility, with MICs of 0.12 to 0.5 g/ml; and resistant groups, with MICs of 4 g/ml, 8 g/ml, and 16 g/ml (Table 3). The isolates with reduced susceptibility to ciprofloxacin were resistant to nalidixic acid (MICs of 128 to 256 g/ml), and the trait of resistance was associated with a mutation in gyrase subunit A (S83F, S83Y, or D87N) and enhanced activity of the efflux pump for nalidixic acid. The isolates with a ciprofloxacin MIC of 4 g/ml were highly resistant to nalidixic acid (MICs of ⱖ256 g/ml), and the resistance was associated with a mutation (S83Y) in gyrase A, the presence of qnrS, and enhanced activity of the efflux pumps for ciprofloxacin and nalidixic acid. The isolates with ciprofloxacin MICs of 8 and 16 g/ml also displayed high nalidixic acid resistance (MICs of ⬎256 g/ml), carried two mutations (S83F and D87G) in gyrase A and one mutation (E92K) in topoisomerase IV, and showed enhanced efflux pump activity for nalidixic acid. The isolates with a ciprofloxacin MIC of 16 g/ml also showed enhanced efflux pump activity for ciprofloxacin.
Chiou et al.
ACKNOWLEDGMENTS This work was supported by a grant (DOH98-DC-2021) from the Centers for Disease Control, Department of Health, Taiwan, a grant (MOHW103CDC-C-315-000801) from the Ministry of Health and Welfare, Taiwan, an intramural grant (IDPP03-014) from the National Health Research Institutes, and a grant (H17-Shinkou-Ippan-019) from the Ministry of Health, Labor and Welfare, Japan. We sincerely thank The International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), for kindly providing S. Typhi isolates for this study. We declare that there are no conflicts of interest regarding the publication of this paper.
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