A prospective cohort study was conducted at the travel clinic at the Leiden University Medical Center and at the Hollands Midden Municipal Health Services in Leiden, the Netherlands. During March–September 2011, all adults who made an appointment for travel advice and had the intention to travel to areas outside Europe, North America, and Australia were invited to participate in the study. Travelers <18 years of age and those who traveled >3 months were excluded. Only 1 person in a couple or travel group was included.

Participants were asked to complete an electronic questionnaire and to deliver a rectal swab sample immediately before and immediately after travel. Questionnaires were used to collect demographic data, previous medical history, and travel information. Travelers who acquired MDR-E after foreign travel were asked to fill out a third questionnaire and deliver a third rectal swab 6 months after return.

If travelers were positive for MDR-E 6 months after return, their household contacts were also requested to submit a rectal swab and questionnaire. Household contacts were defined as persons who shared the same household with a participant on a regular basis. MDR-E–positive participants were asked to deliver a fourth rectal swab at the same time. The study was approved by the Leiden University Medical Center medical ethics committee.

Rectal swab samples were collected with Stuart Agar Gel Medium Transport Swabs (Copan Diagnostics, Corona, CA). The swabs were saturated in trypticase soy broth supplemented with cefotaxime 0.25 mg/L and vancomycin 8 mg/L (MP products, Groningen, the Netherlands) and incubated for 24 hours at 37°C. After overnight incubation, the trypticase soy broth samples were subcultured on chromogenic ESBL screening agar (ESBL-ID; bioMérieux, Marcy-l’Étoile, France) and sheep blood agar as a growth control. All gram-negative rods growing on the ESBL-ID were identified by using MaldiTof-MS with BioTyper software version 3.0 (Bruker Daltonics, Breman, Germany), and antimicrobial drug susceptibility testing was performed by using the VITEK2 system (BioMérieux). All isolates underwent ESBL confirmatory disk testing by disk diffusion for ceftazidime and cefotaxime or cefepime (in cefoxitin-resistant isolates), with and without clavulanic acid, as recommended by Clinical and Laboratory Standards Institute guidelines (www.clsi.org).

MICs for meropenem and ertapenem were determined by using Etests (AB Biodisk, Solna, Sweden) according to the manufacturer’s instructions. MICs were interpreted by using EUCAST criteria (www.eucast.org/clinical_breakpoints/).

Molecular characterization of the β-lactamase genes in ESBL-E was performed by using Check-MDR CT103 version 1.1 (Check-Points B.V., Wageningen, the Netherlands) to test microarrays. The principals of the microarray system and interpretation software have been described (7). Concisely, the system combines ligation-mediated amplification with the detection of amplified products on a microarray to detect the various carbapenemase genes: OXA-48, NDM-1, IMP, VIM, and KPC; CTX-M groups: CTX-M group 1, 2, 9 or combined 8/25; and the most prevalent ESBL-associated single-nucleotide polymorphisms in TEM and SHV-variants. Furthermore, the 6 plasmid-mediated AmpC β-lactamases can be identified (www.lahey.org/studies).

Multilocus sequence typing (MLST) was performed on all E. coli isolates by using 7 housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) to determine the corresponding sequence type (ST) and to designate the sequence type complex (STC) by using the MLST Databases at the Environmental Research Institute, University College Cork website (http://mlst.ucc.ie/mlst/dbs/Ecoli).

A logistic regression model was used to determine risk factors for the acquisition of ESBL-E/CP-E after foreign travel for a total of 338 participants. Associations between acquiring an ESBL-E/CP-E after travel and different variables are calculated as odds ratios and p-values. Participants who were positive for ESBL-E/CP-E before travel were analyzed separately. Database processing and statistical analyses (univariate and multivariate analysis) were performed by using the SPSS software version 20.0 (SPSS Inc., Chicago, IL, USA). MLST analysis was performed by using BioNumerics software v.6.6 (Applied Maths, St-Martens-Lathem, Belgium).

In total, 521 travelers were invited to participate in the study; 370 travelers completed 2 questionnaires and sent in 2 rectal swabs and were included in the analysis (Figure 1). The median age of the study population was 33 years (range 19–82), and 234 (63.2%) were women. The median length of stay abroad was 21 days (range 6–90 days). The most common reason for travel was vacation (n = 277).

Of the 370 participants, 113 (30.5%) whose pretravel swab samples were negative acquired MDR-E during foreign travel. Of these 113 participants, 19 (16.8%) still carried MDR-E 6 months after return. In 32 of the 370 participants (8.6%), MDR-E was identified before travel. Twenty (62.5%) of these 32 participants returned with MDR-E, 7 (35.0%) of whom were still colonized after 6 months. No MDR-E was found before or after travel in 225 (60.8%) participants.

For the analysis of travel-associated risk factors, data for 338 participating returning travelers with negative pretravel rectal swab sample test results were used (Technical Appendix Table 1). In total, 65 countries were visited; these are subdivided in 10 subcontinents. The most common destinations were Indonesia (n = 62), Thailand (n = 30), Malaysia (n = 27), Cambodia (n = 21), People’s Republic of China (n = 39), Kenya (n = 30), Tanzania (n = 24), Surinam (n = 20), and South Africa (n = 19).

The highest ESBL-E acquisition rates were identified among participants who visited countries in Asia: 73% in South Asia and 67% in East Asia. Univariate and multivariate analyses showed that the travel destinations South and East Asia were significant risk factors for the acquisition of ESBL-E (p<0.001). Participants traveling to Asia (all subcontinents) were more likely to return with ESBL-E colonization after a self-arranged trip (odds ratio 1.7; p = 0.07) or if they stayed in hostels/lodges (odds ratio 1.9; p = 0.08), although this finding was not statistically significant. There were no other risk factors for the acquisition of ESBL-E after foreign travel. The incidence proportions of ESBL-E after foreign travel are listed in Table 1.

A total of 133 participants were colonized with MDR-E after travel. This group consisted of 113 travelers who had initially negative pretravel swab samples. In addition, 20 participants who had positive pretravel samples also returned colonized with MDR-E. The ESBL-E of these 133 post-travel swab samples consisted of 146 E. coli, 10 K. pneumoniae, and 2 Enterobacter cloacae isolates.

No carbapenemase-producing MDR-E were found among the pre-and post-travel isolates. Molecular characterization of the post-travel isolates demonstrated that CTX-M group 1 ESBL (n = 110) predominated (CTX-M-1–like, n = 4; CTX-M-3–like, n = 1; CTX-M-15–like, n = 85; CTX-M-32–like, n = 20), followed by CTX-M group 9 ESBL (n = 42), CTX-M group 2 (n = 2), and CTX-M group8/25 (n = 1). One E. coli isolate carried an SHV-ESBL (238S+240K). In addition, some isolates coproduced plasmid-mediated AmpC β-lactamase, ACT/MIR (n = 1) or CMY-2 (n = 2).

Thirty-four ESBL-E were isolated from pretravel rectal swab samples from 32 participants: 29 (85.3%) samples were positive for E. coli, 4 for K. pneumoniae (11.8%), and 1 for Citrobacter freundii (2.9%). The CTX-M group 1 ESBL (n = 22) comprised (CTX-M–1 like, n = 4; CTX-M-15–like, n = 16; CTX-M-32–like, n = 2); the remaining ESBL isolates belonged to CTX-M group 9 ESBL (n = 8) and CTX-M group 2 (n = 1). Two E. coli isolates carried an SHV-ESBL (238S+240K).

Coresistance to other classes of antimicrobial drugs was common in pre- and post-travel isolates; 67% displayed resistance to trimethoprim/sulfamethoxazole, 36% to ciprofloxacin, 37% to tobramycin, 35% to gentamicin, and 29% to nitrofurantoin. All isolates were susceptible to colistin and carbapenems.

MLST of 146 E. coli isolates from the post-travel samples identified 86 different STs; 31 new STs were found. The most prevalent STs were: ST38 (12%; n = 17), ST10 (7%; n = 10), and ST131 (4%; n = 9). The distribution of the CTX-M groups and types and STs is displayed in Figure 2. There was no association between ST and ESBL type, nor were STs associated with specific travel destinations. Pretravel isolates showed a similar diversity of STs, of which 3 were ST131.

Of the 133 participants whose samples were positive for ESBL-E after return, 127 (95.4%) completed the follow-up survey and provided samples after 6 months. ESBL-E was isolated from 26 (20.4%) samples (Table 2). None of these participants reported the use of antimicrobial drugs or were hospitalized during the previous 6 months; none were health care workers, and none reported contact with farm animals. Diarrhea was reported by 7 participants.

Of 113 participants who had initially negative pretravel samples and positive samples immediately after return, 19 (16.8%) were still colonized after 6 months. Of these, 7 participants had samples that were positive for E. coli with the same ST 6 months after return. Nine participants were positive for E. coli and had a different ST 6 months after return; 3 were positive for a different species 6 months after return. Eleven household contacts of 4 MDR-E–positive participants agreed to cooperate and submitted a rectal swab sample. ESBL-producing E. coli was isolated from 2 (18.1%) household contacts, each from different households. The first household contact carried a different ESBL-producing E. coli than the associated traveler before and after the trip. Both isolates carried a CTX-M group 9 enzyme. The second household contact was positive for SHV-ESBL-producing E. coli ST2599. The associated traveler’s samples were positive for E. coli ST617 and ST38 immediately after the trip, K. pneumoniae 6 months after return, and the fourth rectal swab sample was positive for a CTX-M-15–like E. coli ST3363.

Of 20 participants whose samples were positive before and after return, 7 (35.0%) participants were still colonized 6 months after return. Of these 7 participants, 5 carried a similar strain: 2 carried a CTX-M group 9–producing E. coli with an identical ST as before the trip, 2 carried a similar ST but with a different CTX-M group enzyme as before the trip, and 1 participant carried a CTX-M group 1–producing K. pneumoniae during the study period; 2 participants returned with E. coli with a different ST. No household contacts were included in this subgroup of travelers.