Two hundred twenty-five elementary and secondary school students and 11 teachers were spending a week (June 11–15, 2001) at a summer camp in Palafrugell, Girona (Spain), when an outbreak of gastroenteritis began on June 14. An epidemiologic investigation involving 200 campers and staff failed to identify the source of the outbreak. Clinical and epidemiologic studies were initiated at the onset of the outbreak, but our participation as reference laboratory began later. Consequently, a limited number of stools from symptomatic patients and related persons were available to us for analysis.

From June 16 to 19, stool samples from 22 ill campers were collected for analysis. On July 6 (18–19 days later), additional stool samples were collected from four ill campers and from 10 asymptomatic household members of the four ill campers. Stool samples were also collected from three asymptomatic food handlers, on June 19, 22, and 28.

Stool suspended in saline was added to trypticase soy broth containing 2 mg/L cefotaxime (TSB-CTX), which after 18 h of incubation at 35°C, was added to similarly supplemented MacConkey broth (MacC-broth-CTX). After an 18-h incubation, a loopful of this broth was spread on similarly supplemented MacConkey agar. A colony of each distinct morphotype was analyzed further.

Stool samples were processed as for isolation of ESCREC, but after the initial growth the MacC-broth-CTX was added to selenite broth. After 8 h of incubation at 35°C, this broth was spread on cefotaxime-supplemented xylose-lysine-deoxycholate agar. Colonies suspected of representing Salmonella were analyzed further.

Eight unprepared food items from the camp kitchen were analyzed for Salmonella and ESCREC as described for stool samples, except that the initial TSB-CTX was replaced by peptone yeast extract broth.

Disk diffusion susceptibility testing to 28 antibiotics (Table) was performed according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines (23). The activity of cefotaxime and ceftazidime, combined with clavulanate, was determined by E test (AB Biodisk, Solna, Sweden). MICs to β-lactam antibiotics were determined by broth microdilution (Sensititre, Trek Diagnostic Systems LTD, West Sussex, England), according to NCCLS guidelines (24,25).

Filter matings were performed with ESCREC donors, using as recipients either E. coli HB101 (Nal Kan) for which transconjugants were selected, according to described methods (26), or the S. enterica isolate from patient P12, for which transconjugants were selected by adding the filter containing mixed growth of E. coli and Salmonella to MacC-broth-CTX. This broth, which was subsequently processed as described above for selection of resistant S. enterica.

Crude extracts of β-lactamases were obtained by ultrasonication. Analytical IEF was performed as previously described (27).

The bla_TEM, bla_CTX-M-9 and bla_CMY-2 genes were amplified as previously described (28–33). The DNA sequence was directly determined from PCR products for both DNA strands (34).

The biotype, as determined for 12 metabolic reactions, was expressed as a 4-digit code (35). The serotype and phage type of Salmonella isolates were determined in the Servicio de Enterobacterias del Centro Nacional de Microbiología, Instituto Carlos III, Majadahonda, Spain (M.A. Usera and A. Echeita). The serogroup of the ESCREC was determined at the Laboratorio de Referencia, Lugo, Universidad de Santiago de Compostela (J. Blanco).

Genomic profiles were analyzed by PFGE with XbaI (Amersham Biosciences UK Limited, Buckinghamshire, England) (36,37). Isolates exhibiting indistinguishable PFGE profiles were considered to represent the same clone.

Plasmid DNA was isolated by using a commercial kit (QIAGEN, Inc., Valencia, CA) and subjected to 0.8% agarose gel electrophoresis both without digestions and after cleavage with PstI, SmaI, or HincII (Amersham Biosciences). For southern hybridization (38), both total and restricted plasmid DNA were transferred to nylon membranes and hybridized with PCR-generated probes for bla_CTX-M-9 (850 bp) and bla_CMY-2 (1,017 bp), as labeled and detected using the ECL^TM Direct Nucleic Acid Labeling and Detection System (Amersham Biosciences).

Comparisons of proportions were tested by using the Fisher exact test (two-tailed).

The 225 student campers, 10–16 years of age, and 11 teachers were from three schools in three cities, Tarragona (T), El Prat (EP), and Sant Boi (SB). Of the 200 campers and staff interviewed, 109 (54.5%), including 3 teachers, had symptoms of gastroenteritis, with no significant differences between the three schools (57%, 49%, and 62% respectively). The most frequent symptoms were abdominal pain (80%), diarrhea (79%), and headache (64%). Two students were admitted to hospital, but after supportive therapy were discharged within 48 hours. No person received antibiotic therapy.

Although stool samples collected from the three (asymptomatic) food handlers were negative for enteropathogens, two of these persons were carriers of ESCREC. One had CTX^R-FOX^S-ESCREC at the second sampling, whereas the other had CTX^R-FOX^R-ESCREC at both the second and third samplings.

In contrast, neither enteropathogens nor ESCREC were detected in stool samples from 10 healthy household members of four ill campers who had both ESCREC and salmonellae in their acute-phase stool sample (for prevalence of ESCREC among household members vs. campers or food handlers, p=0.03 and p=0.04, respectively).

The eight cultured camp food items yielded neither S. enterica serovar Enteritidis nor ESCREC.

In the 11 E. coli isolates phenotypically suspected of ESBL production, a β-lactamase with an isoelectric point of 8.0 was detected. PCR with bla_CTX-M-9-specific primers and sequence analysis confirmed the presence of the CTX-M-9 β-lactamase (not shown).

Three of these isolates had an additional β-lactamase with pI 5.4, which in IEF reacted with penicillin but not with ceftriaxone. PCR with bla_TEM-specific primers confirmed the presence of a TEM-1-like β-lactamase (Table).

All four isolates phenotypically suspected of AmpC β-lactamase production were PCR-positive with ampC-family primers. Sequence analysis confirmed the presence of the CMY-2 β-lactamase (not shown).

Collectively, the 15 ESCREC isolates represented five distinct biotypes (Table). Overall, five serogroups (O20, O55, O86, O145, and non-typable/NT) and five PFGE pulsotypes (A to E) were detected. Biotype, serogroup, and pulsotype corresponded precisely, confirming the presence of five discrete ESCREC clones (Table and Figure 1).

Clone A (CTX-M-9), the most prevalent, was recovered from students from all three cities, EP, SB, and T (Table). Clone B (CMY-2) was recovered from two students from Sant Boi and clone E (also CMY-2) was recovered from a food handler. Clone C (CTX-M-9 + TEM-1) was recovered from both a Sant Boian student and a food handler. Clone D (CTX-M-9 + TEM-1) was recovered from a Sant Boian student.

Plasmid profiles of the ESCREC isolates were largely concordant with clonal assignments (Figure 2A, Table). In all but one isolate, the probes for bla_CTX-M-9 and bla_CMY-2 hybridized to a single large (>150 kb) plasmid (Figure 2).

By Southern blot analysis, the bla_CTX-M-9-containing plasmids (clones A, C, and D) exhibited minor diversity within and greater diversity among clones (Figures 2–4, Table). In the uncut plasmid blot, the large, probe-positive plasmids of clones A and D were uniform in size and larger than those of clone C (Figure 2B). The corresponding PstI Southern blot showed three different patterns, which were not clone-specific (Figure 3, Table). All of these isolates exhibited a band at ~1,289 bp (Figure 3B), whereas three isolates (all clone A) also exhibited a variably sized larger band (Figure 3B, Table). The corresponding SmaI blot showed five patterns among the CTX-M-9-positive isolates, including three closely related patterns among the clone A isolates and unique patterns each for the clone C and D isolates (Figure 4, Table).

In contrast, the bla_CMY-2-containing isolates (clones B and E) were indistinguishable in both the uncut plasmid blot (Figure 2C) and the PstI and HincII blots, which showed a homogeneous single-band pattern for all three isolates (not shown).

One CTX-M-9-positive isolate transferred its resistance by in vitro conjugation to E. coli HB101 but not to a S. enterica isolate from a patient. In contrast, a CMY-2-positive isolate was successfully conjugated with this S. enterica isolate but not with E. coli HB101 (probably due to the donor strain’s production of a bacteriocin that inhibits HB101; data not shown).