We investigated 31 S. Typhi strains that were associated with travel to six different countries. Ribotyping with EcoRI and PFGE with XbaI segregated the isolates into 10 and 22 profiles, respectively. Sixteen strains were resistant or intermediately resistant to at least one antimicrobial, and five showed MDR profiles. Twelve of the resistant strains were NAL resistant, all of which had a nucleotide alteration at either Ser-83 or Asp-87.
PFGE was more discriminatory than ribotyping, as observed previously (13
). Strains from diverse countries, isolated in different years, and with different resistance profiles were identical by ribotyping (), whereas only the isolates sharing geographical linkage, year of isolation, and resistance profiles were identical by XbaI PFGE, with one exception (). This demonstrates that PFGE clusters strains into epidemiologically meaningful groups compared to ribotyping, as reported previously (13
). One exception to this was the identical pulsotypes of strains CBD 1267 and CBD 1346 (). While the two strains had identical ribotypes and PFGE and antibiotic resistance profiles, they were linked to Florida (isolated in 2007) and Pakistan (2008), respectively. An apparent association between the two isolates was not supported by the epidemiological data. Strain CBD 1267 was not associated with international travel and was isolated in a different year than CBD 1346, suggesting that either the patient was not truthful about his/her travel history or that he/she came in close contact with an asymptomatic carrier. However, we have no further information regarding an epidemiological investigation of the patient's exposure history.
PFGE segregated some of the strains that were identical by ribotyping and vice versa ( and ). Considerable genetic heterogeneity was revealed among the isolates since different clones coexisted within the same geographical location. In contrast, clones were distributed among multiple countries. This clonal nature of the S. enterica
serovar Typhi was especially evident by ribotyping (, cluster B). Generally, only strains sharing the same travel history, year of isolation, and resistance patterns were identical by PFGE. However, PFGE also grouped certain strains with different travel histories at a very high similarity (). This pattern of global clonal distribution of strains is consistent with other studies employing SNP analysis, MLST, or PFGE, where diverse strains were identified in the same geography, and clones were distributed in various locations (3
MDR strains were discriminated well by PFGE. The 10 two-drug (NAL-CIP)-resistant isolates were distributed among all the clusters by both ribotyping and PFGE. By ribotyping these isolates showed similarity ranging from 26% to 100% with either the susceptible or the MDR strains (). Interestingly, none of these NAL-CIP-resistant strains had identical pulsotypes with either susceptible or MDR strains although they clustered at high similarity by PFGE. Le et al. have suggested that there was a clonal expansion of MDR S
. Typhi, but the replacement of classical first-line antibiotics with fluoroquinolones resulted in loss of the MDR phenotype and an increase in NAL resistance in strains isolated in Vietnam (17
). In a comparison study, these investigators have demonstrated that several different PFGE profiles were present in the H-58 haplotype. Our findings agree with the above concept of microevolution within S
. Typhi clones based on our PFGE genetic relatedness data among MDR, NAL-CIP-resistant, and fully susceptible isolates. NAL-CIP resistance, although present in 10 of 12 strains associated with the Indian subcontinent, was not limited to that region since a NAL-CIP-resistant strain in this study was related to travel to Peru, and a CIP-resistant strain was from Haiti. However, the fact that insufficient numbers of strains were investigated from other regions needs to be considered. The recent global increase in NAL-CIP-resistant strains is likely due to increased use of fluoroquinolones in the 1990s, leading to selection of resistant strains (33
). The NAL-resistant H-58 haplotype has undergone a recent clonal expansion (14
). It is also likely that MDR strains belonging to the H-58 haplotype have recently lost their MDR plasmid, causing a decrease in the incidence of MDR strains with concomitant replacement by NAL single drug resistance, as shown in a previous study (17
). A comparison of PFGE profiles and a complete SNP analysis will provide more information about the nature of the strains in our study and illustrate if they belong to the H-58 haplotype.
Three of the MDR strains had a 750-bp integron with the dfr7
gene cassette conferring resistance to trimethoprim, as seen previously (18
). Though resistant to SXT, two out of five MDR strains did not harbor integrons with the dfr
gene (). While we did not screen the isolates for plasmids, a plasmid-integron association is highly possible, as noted previously (28
). The fact that the majority of the resistant isolates (~93%) had intermediate resistance to CIP regardless of whether they were resistant to NAL suggests that NAL resistance is not a good indicator of DCS, which is consistent with a previous study (7
). In this study, the fact that strains with no resistance to any drug have demonstrated DCS is alarming because of the longer clearance times and treatment failures associated with DCS (7
). We have examined a NAL-susceptible yet otherwise resistant strain (, CBD 1350) with DCS for the presence of point mutations in the gyrA
gene. This strain had no mutations in the two regions investigated. The basis of the apparent DCS could be mutations either in other regions of gyrA
which we have not examined or in other genes including gyrB
. Since this strain is resistant to other drugs, including AMP, CHL STR, and SXT, decreased membrane permeability or increased efflux pump action may have been involved in the DCS. This may also apply to other NAL-susceptible MDR isolates with intermediate resistance to CIP. The greater number of SNPs encoding Phe-83 than Tyr-83 in the gyrA
QRDR observed in this study is consistent with previous studies (10
Our molecular typing data shed light on the distribution patterns and the dominant S. Typhi types circulating throughout the globe. Twenty strains had the ribotype R6, making it the predominant type in this study. Haitian strains are equally diverse, similar to the Indian subcontinent strains, demonstrating that Haiti is an important reservoir of the organism. Various clones have been shown to be circulating and causing infections, implying that these types are maintained in the population. Since carriers are important reservoirs of this organism, screening and treatment of carriers in areas of endemicity will likely be very helpful in preventing spread of this disease, thus reducing the burden of disease.
Recent isolates (isolated in 2010) from our study are predominantly resistant to NAL-CIP and appear to have lost their MDR properties, as previously observed (17
). Antibiotic recycling by reusing traditional first-line drugs (e.g., tetracycline, chloramphenicol, and trimethoprim) should be carefully considered. Clinicians in the United States need to be aware of the resistance of this organism to various antimicrobials, especially those associated with the Indian subcontinent, and consider azithromycin or ceftriaxone for empirical treatment (8
). Continued monitoring of resistance patterns is essential for successful treatment of travel-related bacterial infections.
Approximately 51% of the cases in this study were in patients who were ≤18 years old, indicating that high-risk groups—especially children—need to be vaccinated and to take extra precautions regarding food and water that are consumed while traveling. The fact that the majority of the patients were unvaccinated also supports the need for expanded public awareness of vaccine availability and efficacy. Three patients in our study were food handlers, and one was a health care worker. Following foreign travel, given the nature of their work, these groups could represent heightened risk to public health. With the increased globalization of the food supply, and the large number of travelers to regions of endemicity, typhoid is not only a problem for developing countries but also for developed countries. An increased focus in regions of endemicity on improving sanitary conditions, the quality of food and water, identification and treatment of carriers, and surveillance for disease and an effective immunization program will all help reduce the incidence and burden of disease.
Our study represents 3 years of strain and epidemiological data associated with multiple countries of origin and elucidates typing and resistance patterns of travel-related S. Typhi in Florida. Strains isolated in the year 2009 were not available for study, which was a minor deficiency of our study. To our knowledge, this is the first report about the use of pyrosequencing technology to analyze the gyrA gene QRDR in S. Typhi. Our study is consistent with other studies and reaffirms the global clonal distribution and the increase in the NAL resistance of this very important organism.