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Journal of Clinical Microbiology, March 2005, p. 1205-1209, Vol. 43, No. 3
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.3.1205-1209.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Analysis of Salmonella enterica Serotype Typhi Pulsed-Field Gel Electrophoresis Patterns Associated with International Travel

K. Kubota,¹ T. J. Barrett,^1* M. L. Ackers,¹ P. S. Brachman,² and E. D. Mintz¹

Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention,¹ Rollins School of Public Health, Emory University, Atlanta, Georgia²

Received 19 August 2004/ Returned for modification 29 September 2004/ Accepted 8 November 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Typhoid fever is a significant cause of morbidity and mortality worldwide, causing an estimated 16 million cases and 600,000 deaths annually. Although overall rates of the disease have dramatically decreased in the United States, the number of travel-related infections has increased in recent decades. Drug resistance among Salmonella enterica serotype Typhi strains has emerged worldwide, making antimicrobial susceptibility testing an important function in public health laboratories. Pulsed-field gel electrophoresis (PFGE) subtyping of food-borne and waterborne pathogens has proven to be a valuable tool for the detection of outbreaks and laboratory-based surveillance. This retrospective study examined the distribution of PFGE patterns of S. enterica serotype Typhi isolates from patients with a history of international travel. Isolates were collected as part of a passive laboratory-based antimicrobial susceptibility surveillance study. Isolates were PFGE subtyped by using the restriction enzyme XbaI to restrict the total genomic DNA. Isolates indistinguishable with XbaI were further characterized using the restriction enzyme BlnI. A total of 139 isolates were typed, representing travel to 31 countries. Restriction fragment patterns consisted of 14 to 18 fragments ranging in size from 580 to 40 kbp. Seventy-nine unique PFGE patterns were generated using XbaI. Isolates from the same geographic region did not necessarily have similar PFGE patterns. Of the 139 isolates, 46 (33%) were resistant to more than one antimicrobial agent (multidrug resistant [MDR]). Twenty-seven (59%) of 46 MDR isolates had indistinguishable PFGE patterns with both XbaI and BlnI. It appears that MDR S. enterica serotype Typhi has emerged as a predominant clone in Southeast Asia and the Indian subcontinent.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Typhoid fever infections caused by Salmonella enterica serotype Typhi are a significant cause of morbidity and mortality among children and adults in developing countries. The World Health Organization estimates that 16 million illnesses and 600,000 deaths worldwide are due to S. enterica serotype Typhi infections annually (4). Infections due to S. enterica serotype Typhi result in bacteremia characterized by remittent fevers, headache, malaise, abdominal discomfort, constipation or diarrhea, and, in some cases, a characteristic "rose spot" rash (19). Approximately 3 to 5% of patients with acute typhoid fevers may become asymptomatic chronic carriers of S. enterica serotype Typhi (15). The majority of infections result from consuming foods or water contaminated by feces of patients or carriers. Public health prevention measures include purification of water supplies, sewage control, treatment of chronic carriers, and sanitary and hygiene education especially among food handlers. In the United States, persons at highest risk for acquiring typhoid fever are travelers to countries where S. enterica serotype Typhi is endemic (1, 8, 12, 21, 23).

Several antimicrobial agents are used for treating typhoid fever and asymptomatic carriers. However, emerging multidrug resistance among S. enterica serotype Typhi strains has compromised treatment and led to recommendations for more prudent use of these antimicrobial agents. In the 1990s reports of strains having multiple resistance to chloramphenicol, ampicillin, streptomycin, sulfonamides, trimethoprim, and tetracyclines emerged worldwide, especially from countries in Southeast Asia, Africa, and the Indian subcontinent, where S. enterica serotype Typhi is endemic (22).

Pulsed-field gel electrophoresis (PFGE) has been used extensively as a molecular tool for subtyping S. enterica serotype Typhi strains. Several reports have demonstrated the utility of PFGE for surveillance and investigations of S. enterica serotype Typhi and other Salmonella serotypes (10, 18, 24, 27). However, these reports have been limited to the study of local epidemics or to surveillance of drug-resistant strains in a particular country or geographic region.

Despite the growing trend of antimicrobial resistance among S. enterica serotype Typhi strains worldwide, little is known regarding the prevalence and distribution of drug-resistant S. enterica serotype Typhi strains in residents of the United States. In 1996 to 1997, the Centers for Disease Control and Prevention (CDC) conducted active laboratory-based national surveillance for S. enterica serotype Typhi. During the study period, antimicrobial susceptibility testing was performed on all strains from both domestically acquired and travel-associated cases. The objectives of this study were to investigate the extent of genetic diversity among S. enterica serotype Typhi isolates from patients with travel-associated cases and to determine whether multidrug resistance in S. enterica serotype Typhi was associated with the spread of one or more bacterial clones. The results of the study were expected to determine whether PFGE of S. enterica serotype Typhi might be used to (i) determine the likely geographic origin of typhoid fever cases for patients without a clear travel history and (ii) monitor the geographic spread of multidrug-resistant (MDR) S. enterica serotype Typhi.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolates. The CDC initiated active laboratory-based surveillance for typhoid fever from June 1996 to May 1997 to determine the prevalence and distribution of antimicrobial-resistant S. enterica serotype Typhi in the United States. Patient isolates and questionnaires were obtained from 45 state and territorial public health laboratories as previously described (1). S. enterica serotype Typhi isolates were stored in defibrinated sheep blood in liquid nitrogen (Lampire Biological Laboratories, Pipersville, Pa.). For PFGE, S. enterica serotype Typhi isolates were grown overnight at 37°C on Trypticase soy agar plates supplemented with 5% sheep blood (Becton Dickinson, Cockeysville, Md.). A total of 139 Salmonella serotype Typhi clinical isolates from patients with a history of international travel were selected for this PFGE subtyping study. Isolates from patients with a history of travel to more than one country were excluded from this study.

Antimicrobial susceptibility testing. All isolates were tested at the CDC as previously described (1). The standard disk diffusion method was used to determine susceptibility to 12 antimicrobial agents: ampicillin, chloramphenicol, ciprofloxacin, trimethoprim-sulfamethoxazole, gentamicin, kanamycin, nalidixic acid, tetracycline, sulfisoxazole, ceftriaxone, amoxicillin-clavulanic acid, and streptomycin. Interpretive categories (susceptible, intermediate, and resistant) for zones of inhibition surrounding various antimicrobial agent-containing disks were determined using interpretive criteria of the National Committee for Clinical Laboratory Standards (17). Since ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole have traditionally been the drugs most frequently used to treat typhoid fever, S. enterica serotype Typhi isolates resistant to these three drugs were defined as MDR S. enterica serotype Typhi (MDRST).

PFGE. PFGE was performed using the PulseNet 1-day standardized protocol with modifications (20). Bacterial cells were suspended in cell suspension buffer (100 mM Tris, 100 mM EDTA, pH 8.0), and the cell density was adjusted to a turbidity reading of 0.48 to 0.54 (Dade Behring Microscan turbidity meter; West Sacramento, Calif.). Proteinase K was added to a final concentration of 1 mg/ml, and 500 µl of cell suspensions was added to 500 µl of 1% SeaKem agarose (FMC, Rockland, Maine). Three hundred microliters of the agarose mixture was pipetted into reusable plug molds (Bio-Rad Laboratories, Hercules, Calif.). Solidified agarose plugs were transferred to a tube containing 5 ml of lysis buffer (50 mM Tris, 50 mM EDTA, 1% Sarkosyl [pH 8.0]) and 25 µl of proteinase K (20 mg/ml) incubated in a shaking water bath at 54°C for 2 h. Plugs were washed two times with type 1 water for 15 min each time and four times with 0.01 M Tris-EDTA (Gibco BRL, Grand Island, N.Y.) for 15 min each time in a shaking water bath.

Agarose-embedded DNA plugs were cut (2.0 mm) and restricted with 50 U of XbaI (Roche Molecular Biochemicals, Indianapolis, Ind.) for 2 h at 37°C. The digested DNA plugs were loaded on the comb, and a 1% SeaKem agarose gel was prepared using 0.5x Tris-buffered EDTA buffer (Sigma, St. Louis, Mo.) and electrophoresed using a CHEF Mapper (Bio-Rad) with switch times of 2.12 to 63.8 s at 6 V/cm for 18 h at 14°C. Gels were stained using ethidium bromide (1 mg/ml) and destained with two deionized water washes. Gel images were obtained using a Gel Doc 1000 imager (Bio-Rad) under UV transillumination.

Analysis of PFGE patterns was performed using Molecular Analyst Fingerprinting Plus software with data sharing tools, version 1.6 (Bio-Rad). A Salmonella serotype Newport strain (AM01144) was used as a reference standard to normalize gels. Dice similarity coefficients and unweighted pair group method with averages were used to calculate similarity coefficients. Isolates with indistinguishable PFGE patterns when restricted with XbaI were further analyzed by restricting DNA plugs with 25 U of BlnI and running the PFGE gel under the same electrophoresis conditions. Each unique PFGE pattern was assigned a pattern name based on the restriction endonuclease, X01 for XbaI and A26 for BlnI.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Travel-associated cases. A total of 139 S. enterica serotype Typhi isolates from patients with travel-associated cases including 46 that were MDR were examined using PFGE. Only those isolates from patients traveling to a single country were included in this study. Ninety-five (68%) of the 139 isolates selected for PFGE subtyping were from typhoid fever patients who had traveled to either Bangladesh, Haiti, India, Mexico, Pakistan, or the Philippines. Travel to these six countries was responsible for approximately 79% of typhoid fever cases diagnosed in the United States between June 1996 and May 1997. The remaining 44 (32%) isolates were from patients who had traveled to 25 other countries.

Subtyping by PFGE with the restriction enzyme XbaI showed that isolates associated with travel to a single country often had different PFGE patterns (Fig. 1). Eleven isolates from patients with cases associated with travel to Bangladesh had 10 unique PFGE patterns, and 10 isolates from patients with cases associated with travel to Mexico each had a unique PFGE pattern. Likewise, among 10 isolates from patients with cases associated with travel to the Philippines, eight had different PFGE patterns, and of six isolates from patients with cases associated with travel to Indonesia four different PFGE patterns were found. Finally, four isolates from patients with cases associated with travel to Nigeria each had a unique PFGE pattern. Different PFGE patterns of isolates from the same country were generally so diverse that pattern clustering was not apparent (Fig. 1).

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FIG. 1. Dendrogram of unique S. enterica serotype Typhi PFGE patterns.

In contrast, some PFGE patterns were seen in more than one country. Isolates from three patients with cases associated with travel to Bangladesh (1), Mexico (1), or El Salvador (1) all had the same PFGE pattern when restricted with XbaI enzyme. XbaI-digested genomic DNA produced 79 unique patterns associated with travel to 31 different countries (Fig. 1). Two isolates from patients with cases associated with travel to India and the two isolates from patients traveling to the Philippines shared a common PFGE pattern which was not seen in any other countries. Twenty-four isolates from patients with cases associated with travel to India had an indistinguishable PFGE pattern that matched the pattern from isolates from patients with cases associated with travel to Pakistan (12), Vietnam (2), and Bangladesh (1). Most isolates with this particular PFGE pattern were MDR; however, four isolates from India and one isolate from Pakistan that had this PFGE pattern did not have an MDR phenotype.

Seventeen additional isolates that represented patients traveling to 17 different countries all had different PFGE patterns. Isolates from patients traveling to either Colombia (2) or Guatemala (2) also had different PFGE patterns. Of two isolates from patients with cases associated with travel to El Salvador, one isolate had a unique PFGE pattern and the other isolate had a pattern which was also seen in Mexico and Bangladesh. Finally, three isolates from travelers to Guyana each had a unique pattern. Although some PFGE patterns were seen in isolates from more than one country, most isolates from different countries had different PFGE patterns. The value for Simpson's index of diversity was 0.952 based on 79 unique PFGE types. This indicated a high degree of strain diversity among these Salmonella serotype Typhi isolates.

Multidrug resistance cases. Forty-six of 139 isolates were defined as MDRST because they demonstrated resistance to at least ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole, three antimicrobial agents that have traditionally been used for treatment of typhoid fever (1). All 46 isolates were also resistant to streptomycin and sulfamethoxazole; 45 were also resistant to tetracycline, and 11 of these were also resistant to nalidixic acid. The 46 MDRST isolates were almost all from travelers to south Asia: 26 (57%) from India, 12 (26%) from Pakistan, 4 (9%) from Vietnam, and 3 (7%) from Bangladesh. One (2%) was from Haiti. Nine different PFGE patterns were seen among MDRST isolates; however, 34 (74%) MDRST isolates had a common PFGE pattern when DNA was restricted with the XbaI enzyme. Twenty (59%) of the isolates with this PFGE pattern were from patients with cases associated with travel to India; 11 (32%) were from patients with cases associated with travel to Pakistan; 2 (6%) were from travelers to Vietnam; and 1 (3%) was from a traveler to Bangladesh. Other MDR isolates from Bangladesh (2), India (6), Vietnam (2), Pakistan (1), and Haiti (1) did not have this common pattern.

MDR isolates having a common XbaI PFGE pattern were further characterized using a second restriction enzyme, BlnI. Twenty-seven (79%) of 34 isolates had a common BlnI PFGE pattern. The 27 isolates sharing a common PFGE pattern when DNA was restricted with both XbaI and BlnI enzymes were defined as belonging to an MDR PFGE clone (Table 1). Eighteen (67%) of 27 MDR isolates from patients with cases associated with travel to India belonged to this clone, as did seven (26%) MDR isolates from Pakistan and two (7%) MDR isolates from Vietnam. None of the three MDR isolates from Bangladesh belonged to this clone, nor did the MDR isolate from Haiti.

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TABLE 1. S. enterica serotype Typhi resistance types by country

In total, three different patterns of antimicrobial resistance to at least five antimicrobial agents were seen among the MDR isolates. Eighteen of 34 isolates that were resistant to six agents (chloramphenicol, trimethoprim-sulfamethoxazole, tetracycline, ampicillin, sulfisoxazole, and streptomycin [CStxTeASSt]) belonged to the MDR clone. Ten were from patients with cases associated with travel to India, six were from patients with cases associated with travel to Pakistan, and two were from patients with cases associated with travel to Vietnam. Among 11 isolates that were additionally resistant to nalidixic acid (CStxTeNaASSt), nine (81%) belonged to the MDR clone. Eight of these nine isolates were from patients with cases associated with travel to India, and one case was associated with travel to Pakistan. Only one case isolate had the resistance type chloramphenicol, trimethoprim-sulfamethoxazole, ampicillin, sulfisoxazole, and streptomycin (CStxASSt). This isolate was associated with travel to India and did not belong to the MDR clone.

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study examined 139 S. enterica serotype Typhi isolates from persons who had traveled to 31 different countries. To our knowledge, this is the first study to examine PFGE patterns of S. enterica serotype Typhi isolates with such geographically diverse origins. Significant strain diversity was seen among isolates from within single countries such as Bangladesh, Pakistan, India, Haiti, Indonesia, Mexico, the Philippines, and Nigeria. Our results support those of previous studies suggesting significant strain diversity among S. enterica serotype Typhi strains in Southeast Asia and the Indian subcontinent (16, 18, 25, 27). Strain diversity was such that geographic clustering of PFGE patterns was not obvious.

Additionally, this study found that some PFGE types were present in geographically distant locations: three isolates from patients who traveled to El Salvador, Mexico, and Bangladesh had the same XbaI PFGE pattern. Thong and colleagues also found similar PFGE fingerprint patterns among S. enterica serotype Typhi isolates from different countries in Southeast Asia (25).

In contrast to the high degree of genetic diversity seen in drug-susceptible S. enterica serotype Typhi isolates, it appears that a predominant strain of MDRST has emerged in India, Pakistan, and Vietnam. Among 46 MDRST isolates, 27 shared an indistinguishable XbaI and BlnI PFGE profile. The relative lack of diversity among MDRST isolates compared with susceptible isolates was previously reported by Fica et al. in outbreaks in Peru and Chile (5, 6). Connerton et al. found different PFGE patterns in isolates from four MDRST outbreaks in Vietnam (3). It is possible that our predominant MDR clone is represented by one of the patterns reported by Connerton et al., but the use of different running conditions precludes accurate pattern comparisons. Another study found similarities in PFGE patterns and plasmid profiles of MDRST isolates from Tajikistan and the Indian subcontinent, suggesting that the ciprofloxacin-resistant strains isolated in Tajikistan originated from the Indian subcontinent (7). Several studies have associated large conjugative plasmids with the presence of the MDR phenotype (2, 5, 9, 13, 14, 26). Our data and those of others thus support the suggestion that multiple-drug resistance in S. enterica serotype Typhi is largely due to the clonal expansion of a limited number of strains that have acquired (presumably) plasmid-mediated drug resistance genes.

The limited number of PFGE types among MDRST isolates compared with susceptible S. enterica serotype Typhi suggests that the MDR phenotype has emerged relatively recently. Liu and Sanderson have shown that genomic rearrangements (mainly due to transpositions) have contributed to the genetic heterogeneity among wild-type S. enterica serotype Typhi strains (11). The lack of diversity among MDRST strains suggests that the MDR phenotype may provide a selective advantage that has allowed these strains to spread relatively unchanged. Alternatively, the lack of diversity may indicate that relatively few strains of S. enterica serotype Typhi are capable of receiving and maintaining conjugative resistance plasmids (5). Continued monitoring of MDRST PFGE patterns for the divergence of existing patterns and the emergence of new patterns many help clarify this issue.

PFGE is widely considered to be the "gold standard" for the subtyping of many bacterial species. Nair et al. found PFGE to be more useful than phage typing in discriminating between S. enterica serotype Typhi strains (16), and PFGE has proven to be a useful tool for investigating local epidemics and for surveillance of sporadic cases (18, 27). In the United States, PulseNet (a national laboratory-based surveillance program) has recently begun using a standardized protocol to include testing of sporadic S. enterica serotype Typhi isolates in the United States (20). PFGE results may be useful in at least two ways.

Approximately 80% of acute typhoid fever cases in the United States are associated with international travel, particularly to the Indian subcontinent and Southeast Asia. The remaining 20% occur in persons who deny a history of recent foreign travel. In persons with no recent travel history or those who visited more than one region where typhoid is endemic, PFGE subtyping of S. enterica serotype Typhi isolates could suggest a specific geographic origin for their infections and help focus the epidemiologic investigation on imported foods, visitors, or family members from specific countries. The diversity of PFGE patterns observed would limit this approach to exact pattern matches and would be complicated by the presence of exact matches in more than one country. In the case of MDRST isolates, the PFGE pattern may suggest a specific resistance profile that might not otherwise be noted, since most public health laboratories do not perform routine antimicrobial susceptibility testing. More importantly, the apparent clonality (by PFGE) of most MDRST strains may provide a means to facilitate the study of the spread of MDRST worldwide. More-discriminating methods may be needed for epidemiologic investigations in which the specific source of a local outbreak is being investigated.

 
Use of trade names is for identification only and does not imply endorsement by the CDC or by the U.S. Department of Health and Human Services.

 
* Corresponding author. Mailing address: Centers for Disease Control and Prevention, 1600 Clifton Rd., Mail Stop C03, Atlanta, GA 30333. Phone: (404) 639-4185. Fax: (404) 639-3333. E-mail: tjb1{at}cdc.gov.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, March 2005, p. 1205-1209, Vol. 43, No. 3
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.3.1205-1209.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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