Travel-associated faecal colonization with ESBL-producing Enterobacteriaceae: incidence and risk factors

J Antimicrob Chemother 2013; 68: 2144 2153 doi:10.1093/jac/dkt167 Advance Access publication 14 May 2013 Travel-associated faecal colonization with ESBL-producing Enterobacteriaceae: incidence and risk factors Åse Östholm-Balkhed 1,2, Maria Tärnberg 3, Maud Nilsson 3, Lennart E. Nilsson 3,Håkan Hanberger 1,2 and Anita Hällgren 1,2 * on behalf of the Travel Study Group of Southeast Sweden 1 Infectious Diseases, Department of Clinical and Experimental Medicine, Facultyof Health Sciences, Linköping University,Linköping, Sweden; 2 Department of Infectious Diseases, County Council of Östergötland, Östergötland, Sweden; 3 Clinical Microbiology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden *Corresponding author. Tel: +46-10-1035964; Fax: +46-10-1034764; E-mail: aniha@imk.liu.se Received 23 January 2013; returned 19 February 2013; revised 30 March 2013; accepted 3 April 2013 Objectives: To study the acquisition of extended-spectrum b-lactamase-producing Enterobacteriaceae (ESBL-PE) among the faecal flora during travel, with a focus on risk factors, antibiotic susceptibility and ESBL-encoding genes. Methods: An observational prospective multicentre cohort study of individuals attending vaccination clinics in south-east Sweden was performed, in which the submission of faecal samples and questionnaires before and after travelling outside Scandinavia was requested. Faecal samples were screened for ESBL-PE by culturing on ChromID ESBL and an in-house method. ESBL-PE was confirmed by phenotypic and genotypic methods. Susceptibility testing was performed with the Etest. Individuals who acquired ESBL-PEduring travel (travel-associated carriers) were compared with non-carriers regarding risk factors, and unadjusted and adjusted ORs after manual stepwise elimination were calculated using logistic regression. Results: Of 262 enrolled individuals, 2.4% were colonized before travel. Among 226 evaluable participants, ESBL-PE was detected in the post-travel samples from 68 (30%) travellers. The most important risk factor in the final model was the geographic areavisited: Indian subcontinent (OR 24.8, P, 0.001), Asia (OR 8.63, P, 0.001) and Africa north of the equator (OR 4.94, P¼0.002). Age and gastrointestinal symptoms also affected the risk significantly. Multiresistance was seen in 77 (66%) of the ESBL-PE isolates, predominantly a combination of reduced susceptibility to third-generation cephalosporins, trimethoprim/sulfamethoxazole and aminoglycosides. The most common species and ESBL-encoding gene were Escherichia coli (90%) and CTX-M (73%), respectively. Conclusion: Acquisition of multiresistant ESBL-PE among the faecal flora during international travel is common. The geographical area visited has the highest impact on ESBL-PE acquisition. Keywords: travel medicine, CTX-M, antibiotic resistance Introduction Antibiotic resistance rates in Gram-negative bacteria are rapidly increasing, especially with regard to resistance to third- and fourthgeneration cephalosporins. 1 This increase, although varying in terms of rate and magnitude in different countries, is recognized worldwide and is mainly due to the production of extendedspectrum b-lactamases (ESBLs) in Enterobacteriaceae conveyed by genes such as bla CTX-M, bla TEM and bla SHV. CTX-M enzymes have rapidly become the most important ESBLs, with increases mainly in CTX-M-15 in many countries during the last decade. 1 Moreover, the acquisition of other resistance mechanisms is common, and multiple resistance is often seen among ESBLproducing Enterobacteriaceae (ESBL-PE). 1,2 Unlike infections caused by other multiresistant pathogens such as methicillinresistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and Pseudomonas spp., infections caused by ESBL-PE are often acquired in the community rather than in a healthcare setting. 3 As infections such as urinary tract infections (UTIs) and sepsis caused by Escherichia coli and other Enterobacteriaceae are thought to be caused by bacteria from the patient s own faecal flora, colonization with ESBL-PE is probably a prerequisite for infection with such bacteria in most cases. Colonization of one individual may, if it persists, spread to other family members. 4 6 Horizontal transfer of resistance genes to other gut pathogens may also occur. In recent years, retrospective studies have identified international travel as a risk factor for infection with ESBL-PE, as well # The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com 2144

Travel-associated faecal colonization with ESBL-PE JAC as for ESBL-PE colonization. 7 9 To date, two prospective studies on travellers have confirmed this. 10,11 Descriptive statistics in both of these studies suggest that ESBL-PE acquisition is associated with travel to the Indian subcontinent and/or gastroenteritis during travel, but with diverging results concerning the use of antibiotics during travel as a risk factor for ESBL-PE colonization. 10,11 However, there have been no assessments of the risk factors that have the highest impact on the travel-associated acquisition of ESBL-PE. The objective of the present study was to investigate colonization by ESBL-PE during travel, with a focus on risk factors associated with ESBL-PE acquisition, and to analyse ESBL-PE acquired during travel with regard to susceptibility to different antibiotics as well as the distribution of ESBL-encoding genes. Material and methods Study design and setting An observational prospective multicentre study was performed in the south-east of Sweden, at the infectious disease departments of three hospitals in the region: Ryhov Hospital in Jönköping, Kalmar County Hospital and Linköping University Hospital. Individuals aged 18 years or older attending the vaccination clinics of the infectious disease departments and planning to travel outside the Scandinavian countries for no more than 3 months were asked to participate. Participants were recruited from 1 September 2008 through April 2009. After written informed consent was obtained, the participants were asked to submit faecal samples and to fill in questionnaires before and after travelling (see Supplementary data, available at JAC Online). In the questionnaires, the participants were asked for their gender, age, previous hospitalization (last 5 years) or travel (last 3 years), relevant medical conditions, antibiotic treatment during the preceding 6 months, ongoing peptic ulcer medication or any medication influencing the immune system, details of their journey (duration, purpose, countries visited), use of oral cholera vaccine before travel, symptoms (diarrhoea, other gastrointestinal symptoms, fever) and use of antibiotics or hospitalization during travel. Samples and questionnaires were sent to the clinical microbiology laboratory and study coordinators at Linköping University for analysis. Individuals who did not submit two complete sets of faecal samples and questionnaires were excluded, as were those who submitted their pretravel sample more than 4 months before the start of their journey or their post-travel sample more than 4 months after returning. Travellers colonized with ESBL-PE before travelling were not included in the analysis of factors associated with travel-acquired ESBL colonization. The Regional Ethical Review Board in Linköping, Sweden, approved the study (Ref. no: M94-08, T109-08). Definitions ESBL-producing Enterobacteriaceae were defined according to Giske et al., including plasmid-mediated AmpC and carbapenemases within the concept of ESBL. 12 Classical ESBL enzymes such as CTX-M, TEM and SHV are designated as ESBL A, plasmid-mediated AmpC as ESBL M and carbapenemases as ESBL CARBA. A participant was defined as a travel-associated (TA) carrier when ESBL-PE was detected in the post-travel faecal sample but not in the pretravel sample. A participant was defined as a non-carrier when no ESBL-PE was detected in either of the samples. The countries visited were categorized into geographic regions: Africa- North (including countries north of the equator), Africa-South (countries south of the equator, including the Horn of Africa), Asia (except the Indian subcontinent), Europe, the Indian subcontinent (Bangladesh, Bhutan, India, Nepal, Pakistan and Sri Lanka), Oceania and Australia, America-North (USA, Canada, Mexico) and America-South (including South America, Central America and the Caribbean). Multiresistance was defined according to Magiorakos et al. (i.e. nonsusceptibility to 1 antimicrobial agent in 3 defined groups). 13 As the present study included antibiotics not used in the definition of Magiorakos et al., some minor modifications regarding the definition of groups were applied in the present study: (i) all b-lactamase inhibitor combinations were regarded as one group; (ii) temocillin and mecillinam (i.e. penicillins with anticipated activity against ESBL-PE) were regarded as one group; and (iii) nitrofurantoin was regarded as a separate group. Sampling and microbiological methods Faecal samples were collected by the participants using nylon-flocked ESwab 480CE (Copan, Brescia, Italy). ESwab eluates the entire sample into 1 ml of modified liquid Amies medium. Faecal specimens were sent by mail to the clinical microbiology laboratory, Linköping University, foranalysis. The dates of sampling and the arrival of samples at the laboratory were recorded. Samples were either cultured on arrival or stored at 2708C before culture. Screening for ESBL was performed in two ways: the faecal sample was spread with the flocked swab onto: (i) ChromID ESBL agar (biomerieux, Marcy l Etoile, France) and (ii) chromogenic UTI agar (bio- Merieux), onto which antibiotic discs (Oxoid, Basingstoke, UK) containing ceftazidime, cefotaxime, piperacillin/tazobactam, cefepime or linezolid were placed. Colonies (one isolate per phenotype, i.e. in terms of colour, slime production etc.) growing on the UTI agar within the expected inhibition zone of any of the cephalosporin and/or piperacillin/tazobactam discs or growing on the ChromID ESBL agar plate were characterized to the species level by conventional typing methods. Phenotypic Etests (bio- Merieux) with ceftazidime/ceftazidime+clavulanic acid, cefotaxime/ cefotaxime+clavulanic acid and cefepime/cefepime+clavulanic acid were used to confirm the classical ESBL phenotype (ESBL A ). Cefotetan/ cefotetan+cloxacillin were used to confirm the AmpC phenotype (ESBL M ). Susceptibility testing MICs were determined using the Etest (biomerieux) according to the manufacturer s instructions. MICs of imipenem, meropenem, ertapenem, amikacin, gentamicin, tobramycin, ceftazidime, cefotaxime, cefepime, piperacillin/tazobactam, amoxicillin/clavulanic acid, temocillin, mecillinam, fosfomycin, nitrofurantoin, tigecycline, ciprofloxacin and trimethoprim/ sulfamethoxazole were determined. For quality control purposes, E. coli ATCC 25922 was used to test each batch of Etests and agar plates. EUCASTclinical breakpoints were used to classify isolates as susceptible (S), intermediate (I) or resistant (R). 14 For temocillin, the lower breakpoints (S 8 mg/l, R.8 mg/l) suggested by the BSAC for infections other than UTIs were used. 15 Molecular methods All isolates with an ESBL phenotype were examined for the presence of bla CTX-M. 16 Isolates not carrying bla CTX-M genes were screened for genes belonging to the bla SHV and bla TEM families. 17,18 Isolates not showing evidence of these three classical ESBL gene groups were screened for the presence of bla AmpC according to a routine multiplex PCR analysis of bla AmpC (bla CIT, bla MOX, bla FOX, bla DHA, bla EBC andbla ACC ) based on a method originally described by Pérez-Pérez and Hanson, as were isolates with an AmpC phenotype. 19 In samples where ESBL M or ESBL A were found in different individual isolates, all ESBL-PE were examined for the presence of both corresponding gene sets. 2145

Östholm-Balkhed et al. Statistical methods The prevalence of carriage before travel was calculated as the percentage of carriers among participants submitting a pre-travel sample. The prevalence of carriage after travel was calculated as the percentage of carriers among participants submitting a post-travel sample, including both pre-travel-positive carriers as well as TA carriers. The difference between the rates of ESBL carriage before and after travel was calculated using McNemar s test, omitting individuals who did not submit a post-travel sample. For the analysis of risk factors for the acquisition of ESBL-PEduring travel, TA carriers were compared with non-carriers. Continuous variables such as age and length of journey were categorized into groups with respect to age (years) and numberof days in order to obtain onlycategorical variables. ORs and 95% CI were calculated using logistic regression in an unadjusted and adjusted analysis. Adjusted logistic regression analysis was reanalysed with a manual stepwise elimination. In the final model, variables were included that either reached significance in the unadjusted (univariate) analysis or in the adjusted analysis with all factors included. We then analysed the influence of each factor byadding oreliminating them one byone, keeping those with either retained significance or a confounding effect. In the final model 12 factors were included. Datawere analysed using the STATA 11.2 software package. P,0.05 was considered statistically significant. Results Study population During the 9 month study period, a total of 262 individuals provided written informed consent and were enrolled. Two hundred and fifty-one participants provided a pre-travel sample and of these 231 provided a post-travel sample; among these 251 pretravel and 231 post-travel samples, ESBL-PE was detected in 6 (2.4%) and 72 (31.2%) participants, respectively (McNemar test: x 2 ¼64.06, P,0.0005). Pre-travel and post-travel samples were submitted at a median time of 15 days (range: 1 114) before departing from Sweden and 3 days (range: 0 191) after return, respectively. Among the 231 travellers who provided a complete set of pre- and post-travel faecal samples and questionnaires (see Supplementary data at JAC Online), five were colonized by ESBL-PE before travel and were excluded from further analysis. The remaining study population (n¼226) consisted of 92 (41%) male and 134 (59%) female participants with a median age of 54 years (range: 18 76 years); further characteristics of this population are provided in Table 1. Fifty-seven countries on all continents except Antarctica were visited, with a median of two visitors per country (range 1 39). Most participants visited only one country, but 22 and 14 travellers visited two and three or more countries, respectively. Eight participants visited more than two continents. The numbers of visits per geographic region are shown in Table 1. The most visited area was Africa south of the equator, with 71 visits; Tanzania (29 visits) and South Africa (16 visits) were the most commonly visited countries in this area. Egypt (23 visits), Thailand (39 visits) and Peru (11 visits) were the most commonly visited countries in Africa north of the equator, Asia and America-South, respectively. In 68 (30%) of 226 participants with negative pre-travel samples, ESBL-PE was detected in the post-travel sample, and these were consequently regarded as TA carriers. In 158 participants, ESBL-PE was not detected in either of the two faecal samples, so these were characterized as non-carriers. The two groups were compared with respect to demographic data and health- and travel-related factors (Table 1). No ESBL-PE was found in samples from travellers to Europe, Australia and Oceania or America-North. Risk factors The results of the univariate analysis are presented in Table 1. Age category, travel to Africa north of the equator, the Indian subcontinent or Asia, symptoms during travel (fever, diarrhoea and other gastrointestinal symptoms), travel, previous 5 years, hospital care, Sweden, previous 5 years and backpacker-style journey were included in the final stepwise adjusted model. The results of this analysis are presented in Table 2. In this analysis, age, travel to Africa north of the equator, the Indian subcontinent or Asia, symptoms during travel (fever, diarrhoea and other gastrointestinal symptoms) remained significant. The most important risk factor for acquiring ESBL-PE during travel was the geographical area visited, with the Indian subcontinent showing the highest risk (OR 24.8, P, 0.001), followed by Asia (excluding the Indian subcontinent) (OR 8.63, P,0.001) and Africa north of the equator (OR 4.94, P¼0.002). Symptoms during travel diarrhoea (OR 2.46, P¼0.029) and other gastrointestinal symptoms (i.e. a number of different gastrointestinal symptoms reported by the participants) (OR 2.99, P¼0.041) also remained significant in the final model, increasing the risk of becoming a TA carrier. By contrast, fever during travel was shown to reduce the risk of colonization (OR 0.20, P¼0.044) in the final model (Table 2). Age also seemed to affect the risk of acquiring ESBL-PE during travel, with a higher risk in all age groups compared with theyoungest travellers (18 34 years) (Table 2). In the final model, the highest risks were found among travellers aged 65 years (OR 7.38, P¼0.007), with lower risks in travellers aged 35 49 years (OR 5.23, P¼0.016) and travellers aged 50 64 years (OR 3.93, P¼0.037). No other variables remained significant in the final model. Microbiological results From the 68 participants with TA colonization, 116 isolates with an ESBL phenotype were recovered and analysed (median: 1, range: 1 6 isolates/person). E. coli was the most commonly found species with 104 (90%) isolates from 61 (90%) TA carriers. Ten isolates (8.6%) of Klebsiella pneumoniae were recovered from 7 (10%) TA carriers. Proteus vulgaris and Enterobacter cloacae were isolated from one TA carrier each. Two participants were colonized with two or more species. From these 116 isolates, 110 ESBL-encoding genes were recovered. CTX-M was found in 85 (74%) isolates from 50 (73%) TA carriers. CTX-M-15-like genes were found in 36 (31%) isolates from 25 (37%) TA carriers and CTX-M-14-like genes were found in 36 (31%) isolates from 23 (34%) TA carriers. CTX-M-27-like genes, CTX-M-53-like genes, CTX-M-1/61-like genes, CTX-M-2-like genes and CTX-M-3-like genes were found in 5, 5, 3, 2 and 1 isolates and TA carriers, respectively. A variety of SHV genes were found in 6 (5%) isolates from 5 (7%) TA carriers. TEM-19 was found in one isolate/participant. Overall, 37 isolates from 26 TA carriers showed the ESBL M phenotype and were screened for genes encoding plasmid-mediated AmpC. CIT-like and DHA-like genes were 2146

Travel-associated faecal colonization with ESBL-PE JAC Table 1. Characteristics of travellers and unadjusted (univariate) risk factor analysis All [n¼226 (%)] TA carriers [n¼68 (%)] Non-carriers [n¼158 (%)] OR (95% CI) P value Demographic and background data male 30 (44) 62 (39) female 38 (56) 96 (61) 0.81 (0.46 1.45) 0.494 age, years (median) 54 54.5 53 18 34 45 (20) 7 (10) 38 (24) 1 35 49 48 (21) 19 (28) 30 (18) 3.56 (1.31 9.59) 0.012 50 64 87 (38) 27 (40) 60 (38) 2.44 (0.97 6.16) 0.059 65 46 (20) 15 (22) 31 (20) 2.63 (0.95 7.25) 0.062 travel, previous 5 years 208 (92) 65 (96) 143 (91) 2.12 (0.59 7.64) 0.250 Previous medical history antibiotic treatment, previous 6 months 29 (13) 6 (9) 23 (15) 0.57 (0.22 1.47) 0.242 hospital care, Sweden, previous 5 years 66 (29) 12 (18) 54 (34) 0.42 (0.21 0.85) 0.016 hospital care, outside Sweden, previous 5 years 3 (1.5) 0 (0) 3 (2) a a diabetes 7 (3) 1 (1.5) 6 (4) 0.38 (0.04 3.18) 0.369 malignancy 4 (2) 1 (1.5) 3 (2) 0.77 (0.08 7.50) 0.819 inflammatory bowel disease 1 (0.5) 1 (1.5) 0 (0) a a chronic urinary tract disease 3 (1.5) 2 (3) 1 (0.5) 4.76 (0.42 53.4) 0.206 medication for ulcers or gastritis 17 (8) 6 (9) 11 (7) 1.29 (0.46 3.65) 0.627 immunomodulating medication 4 (2) 1 (1.5) 3 (2) 0.77 (0.08 7.55) 0.823 Travel-associated data length of journey, days [median (range)] 16 (4 119) 16 (8 62) 16 (4 119) 10 55 (24) 17 (25) 38 (24) 1 11 20 117 (52) 31 (46) 86 (54) 0.86 (0.43 1.72) 0.667 21 54 (24) 20 (29) 34 (22) 1.18 (0.53 2.66) 0.683 Area visited Europe 15 (7) 0 (0) 15 (9) a a Africa, south of equator 71 (31) 15 (22) 56 (35) 0.52 (0.27 0.99) 0.049 Africa, north of equator 30 (13) 13 (19) 17 (11) 1.96 (0.89 4.30) 0.093 Asia (except Indian subcontinent) 58 (26) 26 (38) 32 (20) 2.44 (1.31 4.55) 0.005 Indian subcontinent 14 (6) 10 (15) 4 (3) 6.64 (2.00 22.0) 0.002 Australia and Oceania 2 (1) 0 (0) 2 (1.5) a a America-South 29 (13) 5 (7) 24 (15) 0.44 (0.16 1.22) 0.114 America-North 16 (7) 0 (0) 16 (10) a a Type of journey visit to relatives and friends 28 (12) 9 (13) 19 (12) 1.09 (0.47 2.55) 0.839 business journey 23 (10) 4 (6) 19 (12) 0.45 (0.15 1.37) 0.159 tourist journey 164 (73) 48 (71) 116 (73) 0.83 (0.44 1.56) 0.558 backpacker style 24 (11) 11 (16) 13 (8) 2.11 (0.89 4.98) 0.089 hospital care during journey 1 (0.5) 0 (0) 1 (0.5) a a oral cholera vaccine before journey 111 (49) 37 (54) 74 (47) 1.43 (0.80 2.55) 0.225 Symptoms during journey fever 21 (9) 7 (10) 14 (9) 1.15 (0.44 3.00) 0.771 diarrhoea 95 (42) 36 (53) 59 (37) 1.81 (1.02 3.22) 0.043 other gastrointestinal symptoms 37 (16) 17 (25) 20 (13) 2.23 (1.08 4.59) 0.030 Antibiotic treatment antibiotic treatment after sample 1, before sample 20 (9) 5 (7) 15 (9) 0.75 (0.26 2.16) 0.595 2 of the above, antibiotics taken during travel 16 (7) 5 (7) 11 (7) 1.05 (0.35 3.16) 0.926 a OR could not be calculated due to few or no observations in one of the groups. 2147

Östholm-Balkhed et al. Table 2. Risk factors for TA carriage in the final adjusted logistic regression model after manual stepwise elimination Variable OR (95% CI) P value Age, years 18 34 1 35 49 5.23 (1.36 20.11) 0.016 50 64 3.93 (1.09 14.26) 0.037 65 7.38 (1.71 31.74) 0.007 Area visited Africa, north of equator 4.94 (1.80 13.6) 0.002 Asia (except Indian subcontinent) 8.63 (3.42 21.7),0.001 Indian subcontinent 24.8 (4.98 122),0.001 Symptoms during journey fever 0.20 (0.04 0.96) 0.044 diarrhoea 2.46 (1.09 5.51) 0.029 other gastrointestinal symptoms 2.99 (1.04 8.51) 0.041 Other travel, previous 5 years 4.44 (0.99 20.0) 0.052 hospital care, Sweden, previous 5 years 0.49 (0.21 1.12) 0.093 backpacker-style journey 3.04 (0.70 13.2) 0.136 found in 14 (12%) and 1 (1%) isolates from 12 (18%) and 1 (1%) TA carriers, respectively. In 13 isolates from 12 individuals, no ESBL-encoding genes were found. In three of these participants, other isolates with verified ESBL genes were detected. Thus, among 9 (13%) TA carriers with phenotypic ESBL-producing isolates, no corresponding genes were found. The distribution of genes with regard to the geographic region visited is presented in Figure 1. The number of genes with regard to species and visited geographic region is presented in Table 3. MICs of cephalosporins were high, and only 15%, 10% and 23% of isolates were interpreted as susceptible to ceftazidime, cefotaxime and cefepime, respectively (Table 4). Susceptibility to carbapenems was high (99% 100%). b-lactam/b-lactamase inhibitor combinations exhibited a lower rate of activity as 84% and 73% of isolates were susceptible to piperacillin/tazobactam and amoxicillin/clavulanic acid, respectively. For mecillinam, the range of MICs among isolates was wide; however, the majority (94%) was interpreted to be susceptible. Regarding temocillin, 77% of isolates were interpreted as susceptible. The highest rates of susceptibility to antimicrobial agents other than b-lactam antibiotics were observed for fosfomycin (97%), amikacin (96%), nitrofurantoin (93%) and tigecycline (91%). The activities of gentamicin and tobramycin were low, with only 58% and 53% of isolates, respectively, interpreted as susceptible. Susceptibility rates for ciprofloxacin and trimethoprim/sulfamethoxazole were also low: 66% and 30%, respectively (Table 4). Multiresistance was detected in 77 (66%) isolates from 48 (71%) TA carriers. These isolates showed non-susceptibility to between three and eight groups of antibiotics (median: four), and the most common combination, seen in 43 (56%) isolates, was reduced susceptibility to third-generation cephalosporins, trimethoprim/sulfamethoxazole and aminoglycosides. Regarding the geographical distribution, multiresistance was found in 40%, 66%, 73%, 73% and 80% of isolates (n¼116) from participants who had travelled to Africa-North, Africa-South, Asia, India and America-South, respectively. Participants (n¼68) colonized with at least one isolate with multiresistance with regard to geographical distribution formed 54%, 73%, 73%, 80% and 80% of participants that had visited Africa-North, Africa-South, Asia, India and America-South, respectively. (One person had travelled to both Asia and Africa-South and is consequently counted twice.) Rates concerning America-South should be interpreted with caution owing to the low number of individuals who became TA carriers after travel to this region. Discussion This study confirms that the acquisition of ESBL-PE among the faecal flora during international travel is common. It also demonstrates that, although there are other factors that significantly affect the risk of ESBL-PE colonization, the geographical area visited is the factor with the greatest impact. The colonization rate before travel was 2.4%, consistent with previous findings in Swedish outpatients (2.1% 3.0%). 20 In the limited number of studies addressing the colonization rate among non-hospitalized individuals in different countries, the rate of ESBL-PE carriers varied greatly between geographic regions. Low to modest rates of ESBL-PE colonization were found among healthy individuals in an isolated Amazonian village (8%), healthy volunteers in Japan (6.4%), outpatients in Madagascar (10.1%), elderly people in China (7.0%) and outpatients (15.4%) and healthy volunteers (13.1%) in Saudi Arabia. 21 25 These findings contrast with the very high carrier rates found in Cairo (63.3%) and rural Thailand (58.2%). 26,27 To some extent, these regional differences agree with the results in the present study. For instance, 13 of 30 (43%) visitors to Africa north of the equator acquired ESBL-PE. In this area, Egypt was the most commonly visited country. Likewise, 26 of 58 (45%) travellers to Asia (excluding the Indian subcontinent) acquired ESBL-PE (Table 1), and a majority of these visited Thailand. The rate of TA carriers from different geographic regions may thus to some extent reflect the local colonization rate of the inhabitants. It is worth noting, however, that the duration of the journey did not affect the risk of becoming colonized, which suggests that colonization might occur quite early during travel. As with other bacteria colonizing the gut, the acquisition of ESBL-PE by the ingestion of contaminated food or water (i.e. faecal oral transmission) is thought to be a major transmission route. 5,28 In the present study, as in previous ones, gastrointestinal symptoms and diarrhoea were associated with an increased risk of ESBL-PE acquisition. 10,11 ESBL production has been found in enteroaggregative (EAEC), enteropathogenic (EPEC) and enterohaemorrhagic E. coli (EHEC) that have caused outbreaks of diarrhoea. 29 31 However, when Tängden et al. studied 24 TA ESBL E. coli isolates regarding six genes associated with gastroenteritis, they found only one positive isolate (EPEC). 10 As an analysis of virulence factors was not within the scope of the present study, we do not have data either to support or contradict the findings of Tängden et al., but their conclusions also seem plausible in our setting. We did not find that antibiotic treatment during travel (or between sampling) was associated with the acquisition of ESBL-PE. This contrasts with the results presented by Kennedy et al., who found that 38% of travellers acquiring resistant Enterobacteriaceae (not limited to ESBL-PE) had taken antibiotics during travel, compared 2148

Africa-North CTX-M-3 SHV CTX-M-15 CTX-M-14 ND p-ampc SHV CTX-M-53 CTX-M-27 Asia CTX-M-15 CTX-M-14 Travel-associated faecal colonization with ESBL-PE Indian subcontinent America-South p-ampc TEM ND CTX-M-15 Africa-South CTX-M-14 CTX-M-15 p-ampc ND CTX-M-15 CTX-M-2 CTX-M-14 p-ampc CTX-M-1/61 SHV Percentage TA carriers: 0 % 31 39 % 40 69 % 70 100 % Figure 1. Frequency of TA carriers with respect to geographical area visited and global distribution of ESBL-encoding genes in isolates from TA carriers. JAC 2149

Östholm-Balkhed et al. Table 3. Distribution of detected ESBL-encoding genes in isolates from TA carriers with respect to visited geographic region and species Number of isolates with ESBL-encoding genes per region Gene Africa-North Africa-South Asia Indian subcontinent America-South Total Species (n) CTX-M-15-like 7 4 8 16 1 36 E. coli (36) CTX-M-14-like 13 1 19 3 0 36 E. coli (36) CTX-M-27-like 0 0 5 0 0 5 E. coli (5) CTX-M-53-like 0 0 5 0 0 5 E. coli (5) CTX-M-1/61-like 0 3 0 0 0 3 E. coli (3) CTX-M-2-like 0 0 0 0 2 2 E. coli (2) CTX-M-3-like 1 0 0 0 0 1 E. coli (1) TEM 0 0 0 1 0 1 E. coli (1) SHV 1 1 4 0 0 6 E. coli (2); K. pneumoniae (4) p-ampc (CIT, DHA) 0 6 5 3 1 15 E. coli (14) K. pneumoniae (1) ND 0 3 9 0 1 13 E. coli (4) K. pneumoniae (6) P. vulgaris (1), E. cloacae (1) Total 22 18 55 23 5 123 ND, no gene detected. Table 4. MIC distributions of faecal isolates with ESBL-PE phenotype (n¼116) Antibiotic Number of isolates with MIC (mg/l) for each indicated antibiotic (n¼116) 0.064 0.125 0.25 0.5 1 2 4 8 16 32 64 128.128 %S %R Breakpoints S /R. (mg/l) Imipenem 28 87 1 a 100 0 2/8 Meropenem 113 3 100 0 2/8 Ertapenem 95 10 6 4 1 99 0 0.5/1 Amikacin 11 86 12 2 3 1 1 96 2 8/16 Gentamicin 5 55 3 4 2 4 15 20 4 2 2 58 41 2/4 Tobramycin 23 35 3 2 20 17 9 6 1 53 46 2/4 Ceftazidime 1 4 12 18 17 12 24 16 6 2 4 15 55 1/4 Cefotaxime 2 4 1 2 3 2 2 16 12 16 17 11 28 10 88 1/2 Cefepime 2 9 4 12 15 27 17 20 7 2 1 23 41 1/4 Fosfomycin 1 12 55 29 1 3 7 2 3 3 97 3 32/32 SXT b 20 8 4 3 81 30 70 2/4 Tigecycline 1 19 63 23 8 2 a 91 2 1/2 TZP 1 12 46 23 16 3 1 1 13 84 13 8/16 AMC 27 57 24 6 1 1 73 27 8/8 Temocillin 4 33 52 23 4 77 23 8/8 Mecillinam 1 8 21 31 22 14 6 6 2 1 1 3 94 6 8/8 Nitrofurantoin 1 14 33 29 23 8 3 5 93 7 64/64 Ciprofloxacin b 50 3 19 4 4 2 1 2 31 66 31 0.5/1 AMC, amoxicillin/clavulanic acid; SXT, trimethoprim/sulfamethoxazole; TZP, piperacillin/tazobactam. Breakpoints according to the EUCAST. As no EUCAST breakpoints for temocillin were available, BSAC breakpoints were used. 14,15 a Including one P. vulgaris isolate with intrinsic resistance. b For SXT and ciprofloxacin, 32 mg/l should be interpreted as 32 mg/l owing to the design of the Etest. with 17.3% of non-carriers (P,0.05). 11 However, in the present study, antibiotic usage was lower overall, with corresponding frequencies of 7% and 9%, respectively. This might be explained by differences in antibiotic recommendations, as the use of doxycycline for antimalaria prophylaxis was common in the previous study, but not very common among Swedish travellers. In previous studies, age as a risk factor has been assessed by comparing the median age of TA carriers and controls, and no 2150

Travel-associated faecal colonization with ESBL-PE JAC significant differences were found. 10,11 Likewise, in the present study median ages were very similar between the two groups. However, when dividing the population into different age groups, significant differences with regard to age appeared: the youngest travellers (aged 18 35 years) had a low risk of ESBL-PE acquisition while those aged 65 years had the highest risk. Regarding the distribution of detected ESBL-encoding genes, CTX-M-15-like genes were found among travellers to all continents where ESBL was acquired, emphasizing the global distribution of this enzyme. Similarly, both CTX-M-14-like genes and CIT had widespread distributions (Table 3). By contrast, CTX-M-27- and -53-like genes were only found in Asia, and CTX-M-2-like genes only in America-South. CTX-M-27 was previously regarded as a rather uncommon CTX-M variant, but has in more recent studies from Japan and China been found to be the second and third most common ESBL-encoding gene, respectively, among clinical ESBL-producing Enterobacteriaceae. 32 34 Interestingly, CTX-M-55 (in this study found among the group of CTX-M-53-like genes) has been found to be one of the most commonly encountered ESBL-encoding genes in Enterobacteriaceae from food animals in China and also recently in the UK. 35,36 In the present study, these genes were almost as common as CTX-M-15-like genes among travellers to Asia, suggesting the potential importance of animal-to-human spread via contaminated food. Molecular analysis of ESBL-encoding genes was restricted to bla TEM, bla SHV, bla CTX-M and bla ampc gene groups, which limited the study to some extent. In 13 isolates with an ESBL phenotype no corresponding ESBL-encoding genes could be detected. These isolates might carry other ESBL-encoding genes, such as the OXA-type, or others not included in the analysis. No carbapenemases were found, pointing to another limitation of the study, as the screening was performed with methods mainly targeting cephalosporin-resistant Enterobacteriaceae. Consequently, some carbapenemases with low cephalosporinase activity (OXA-48 for instance) might have escaped detection. The clinical impact of travel-acquired faecal carriage of ESBL-PE is not answered in the present study. However, Laupland et al. found that overseas travel increased the risk of being infected with ESBL-PE (mainly causing UTI) and, as infections of the urinary tract are thought to be caused by bacteria from the patient s own faecal flora, a discussion on the possible clinical impact of our findings may still be justified. 7 Another study showed that the risk of a fatal outcome was 2-fold higher among patients with sepsis caused by ESBL-PE than among patients infected by susceptible Enterobacteriaceae. 37 An important cause of this higher crude mortality is a delay in appropriate empirical therapy in the former group. 37 Assessment of risk factors, including travel history, should therefore be performed when it is suspected that a patient is suffering from Gram-negative sepsis. In order to choose an appropriate empirical therapy it is also crucial to have knowledge of current resistance patterns among ESBL-PE. Multiresistance was common in the present study, but carbapenems still demonstrated good activity. Although carbapenems are regarded as first-line treatment for severe infections when ESBL-PE is suspected, widespread use of carbapenems might lead to an increase in the rate of carbapenem-resistant organisms, so alternative regimens are required. 38 Combination therapy (b-lactam+aminoglycoside) might improve the appropriateness of empirical therapy in septic episodes due to ESBL-PE. 39 While resistance to tobramycin and gentamicin was high (41% 46%) in the present study, only 2% of isolates were resistant to amikacin. These results suggest that amikacin would be the preferred aminoglycoside to use when sepsis caused by ESBL-PE acquired during travel is suspected. In the present guidelines from the Infectious Diseases Society of America for uncomplicated UTIs, nitrofurantoin and pivmecillinam (not available in the USA) are regarded as first-line therapy while trimethoprim/sulfamethoxazole is only recommended when the resistance rate is expected to be,20%. 40 In the present study, susceptibility rates for nitrofurantoin and pivmecillinam were high (93% and 94%, respectively), suggesting that these recommendations can be followed even when ESBL-PE is suspected in afebrile UTI. However, trimethoprim/sulfamethoxazole should not be regarded as a treatment option in this scenario, as 70% of isolates in the present study were resistant. It is also our opinion that a subject s travel history should be obtained before prescribing UTI treatment and, if the subject has travelled overseas recently, urinary culture should always be performed, irrespective of which treatment regimen is chosen, in order to assess resistance. In conclusion, the acquisition of ESBL-PE among the faecal flora during international travel is common and these isolates often show multiresistance. The geographical area visited has the highest impact on ESBL-PE acquisition. Acknowledgements This study was presented in part as an oral presentation at the 20th European Congress of Clinical Microbiology and Infectious Diseases, Vienna, Austria, 2010 and as a poster (S13) at the 27th Annual Meeting of the Scandinavian Society for Antimicrobial Chemotherapy, Stockholm, Sweden, 2010. We are grateful to our colleagues in the Travel Study Group of Southeast Sweden (Liselott Lindvall, Christina Olesund, Helene Jardefors, Per-Åke Jarnheimer, Kerstin Glebe) for assistance in enrolling participants, to Anita Johansson and Anna Ryberg for assisting in the laboratory work and to Mats Fredriksson, LARC, Linköping University, for his advice on the statistical analysis. Funding This work was supported by grants from the Medical Research Council of Southeast Sweden (FORSS-12368, FORSS-36511 and FORSS-87551) and ALF grants from Östergötland County Council (LIO-10885, LIO-16741, LIO-61341 and LIO-127281). Transparency declarations None to declare. Supplementary data The two questionnaires are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/). References 1 Hawkey PM, Jones AM. The changing epidemiology of resistance. J Antimicrob Chemother 2009; 64 Suppl 1: 3 10. 2151

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