CTX-M-15–producing Enteroaggregative Escherichia coli as Cause of Travelers’ Diarrhea

Travelers’ diarrhea is a major public health problem. From patients in whom diarrhea developed after travel to India, 5 enteroaggregative Escherichia coli strains carrying β-lactamase CTX-M-15 were identified; 3 belonged to clonal complex sequence type 38. This β-lactamase contributes to the multidrug resistance of enteroaggregative E. coli, thereby limiting therapeutic alternatives.

the world (4). CTX-M-15 enhances hydrolytic activity against ceftazidime (5). A particular clone of CTX-M-15producing E. coli, characterized by phylogenetic type (phylotype) B2 and sequence type 131 (ST131), seems to be largely responsible for international epidemics of CTX-M-producing E. coli (6). Sequence types (STs) are grouped into clonal complexes by their similarity to a central allelic profi le.
ST131 is a singleton and therefore does not belong to a clonal complex (7). Molecular epidemiologic studies have suggested that the sudden increase in CTX-M-15producing E. coli worldwide was mainly caused by this single clone (ST131) and that foreign travel to high-risk areas, such as the Indian subcontinent, might play a partial role in the spread of this clone across continents (8). The bla CTX-M-15 gene is usually found downstream from the insertion sequence ISEcp1, which may be involved in the clone's dissemination and expression (9). We describe molecular epidemiology and plasmid analyses of 5 CTX-M-15-producing EAEC isolates from patients with travelers' diarrhea who had traveled from Spain to India.

The Study
The study included all patients with diarrhea who visited the Tropical Medicine Unit of Hospital Clinic in Barcelona, Spain, during 2005 and 2006. Patients with diarrhea that started during or shortly after (<5 days) a stay in a developing country were eligible. After the participants provided informed consent, clinical and epidemiologic data were collected.
Among all eligible participants, infection with EAEC and no other enteropathogen was found for 51. Of these 51 EAEC isolates, 5 from patients who had traveled to India were resistant to third-generation cephalosporins. Resistance phenotypes indicated ESBL production. MICs for antimicrobial agents and susceptibility class were determined by using the Clinical and Laboratory Standards Institute breakpoints guideline ( Table 1). All strains were resistant to penicillins; second-, third-, and fourth-generation cephalosporins; and all β-lactamaseinhibitor combinations except piperacillin/tazobactam. Apart from β-lactam susceptibility, the strains showed resistance to other classes of antimicrobial agents, such as fl uoroquinolones, tetracyclines, and monobactams (aztreonam). Positive amplifi cation with specifi c primers and sequencing for the bla CTX-M-15 gene provided positive genotypic confi rmatory test results for ESBL production.
The epidemiologic relationships among the 5 strains were studied by repetitive sequence-based PCR, pulsedfi eld gel electrophoresis, and multilocus sequence typing (10,11). The PCR and pulsed-fi eld gel electrophoresis genomic fi ngerprinting showed that the 5 strains were not epidemiologically related ( Figure 1). However, multilocus  Table 2). E. coli strains were classifi ed into phylogenetic groups by multiplex PCR, described by Clermont et al. (12). The 3 strains in clonal complex ST38 belonged to the potentially virulent phylogenetic group D; the other 2 belonged to group B2 (Table 2).
A PCR method was used to detect genes encoding for typical EAEC virulence factors (2). These genes include aggA and aafA (encoding for adhesions); aap (for dispersin); aatA (for TolC); aggR (for regulation of aggregation); astA, set1A, and sen (for toxins), fyuA (for iron recruitment); agn43 (for antigen 43); and genes encoding for serine protease autotransporter toxins such as pet and sat. Gene aatA was detected in the 5 strains, whereas aap, aggR, and aggA had positive amplifi cation for only 2 of the strains belonging to ST38. The other genes detected are shown in Table 2. EAEC was also identifi ed by typical adherence to HEp-2 cells.
To determine the genetic environment of the bla CTX-M-15 gene, we designed an inverse PCR. We designed the primers by studying the gene sequence and were directed outside the gene. The ISEcp1 insertion sequence was upstream from the bla CTX-M-15 gene, which was also confi rmed by PCR of the specifi c insertion sequence. To confi rm the possible relationship between ISEcp1 and the resistance bla CTX-M-15 gene , we conducted a PCR with the forward primer for the ISEcp1 and the reverse primer for the bla CTX-M-15 gene.
For plasmid extraction of the 5 isolates, we used the method of Kado and Liu (13). Only 3 strains had plasmids ranging from 93 kb to 170 kb (Figure 2, panel A). To confi rm the absence of plasmids in the 2 strains, we conducted S1 digestion of the strains, resolving chromosomal DNA from plasmidic DNA. Southern blot of this digestion showed that the bla CTX-M-15 gene was chromosomally located in these 2 strains, as was the aatA gene (usually found in the plasmid contained in EAEC strains) (data not shown). Finally, the location of the bla CTX-M-15 gene in the 3 plasmid-containing strains was analyzed by using Southern blot from the plasmid extraction. The bla CTX-M-15 gene was located in a plasmid in the 3 strains. The size of the plasmid containing CTX-M-15 varied in each strain (Figure 2, panel B). Plasmids with specifi c known molecular weight were used to provide a range of the size of the plasmids studied.

Conclusions
We identifi ed several features concerning the molecular epidemiology of CTX-M-15-producing EAEC isolates in India. First, all strains belonged to phylogenetic groups D and B2, the 2 groups most commonly found with E. coli infections (14). Second, not fi nding ST131 suggests that ST131 might not be the most common ST among EAEC strains from India and that clonal complex ST38 might play a large role in causing infectious intestinal diseases. Third, the bla CTX-M-15 gene is not only located in the plasmid but may also be in the chromosome. However, previous reports have shown that bla CTX-M-15 is consistently linked with ISEcp1, which means that the chromosomal location might have originated from a previous plasmid location that was part of either a transposon or a cassette within an integron (9). It is also worth noting that the size of the plasmids containing the bla CTX-M-15 gene was not the same in all strains, indicating that this gene may be located in different types of plasmids.