LETTER

It has been shown that Shigella species produce a low-level chromosomally mediated -lactamase which does not appreciably affect the level of resistance to -lactams (2, 10). High rates of resistance to ampicillin among Shigella isolates are due to the production of -lactamases similar to TEM-1 or OXA-1. The OXA-type -lactamase is more prevalent than TEM-1 in ampicillin-resistant Shigella flexneri (2, 6, 7, 8). In Shigella sonnei the most commonly produced -lactamase is the TEM-1 enzyme (2, 6, 7).

We describe here an S. sonnei isolate which was identified by the API ID 32E system (Bio-Merieux) and by antiserum agglutination and which was obtained in 1998 from stool samples of an 8-year-old child hospitalized at Orléans Hospital (Orléans France) for diarrheal disease that appeared after a 2-month stay in Turkey. The strain was resistant to amoxicillin and ticarcillin, either as single drugs or in combination with clavulanate and piperacillin. It was also resistant to trimethoprim-sulfamethoxazole and tetracyclines and susceptible to all cephalosporins, to nalidixic acid, and to fluoroquinolones.

The purpose of the present study was to find the molecular basis of the resistance to amoxicillin-clavulanate in the S. sonnei isolate (UCK strain). A TEM-1-hyperproducing S. sonnei strain (CFS01) and an OXA-1-producing S. flexneri strain (CFS02) were studied as comparators for MIC determinations.

The MICs of amoxicillin, amoxicillin-clavulanate, ticarcillin, ticarcillin-clavulanate (clavulanic acid at a fixed concentration of 2 µg/ml), and cephalothin were determined by dilution in Mueller-Hinton agar (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) with an inoculum of 10⁴ CFU per spot.

The resistance pattern of S. sonnei UCK was characterized by high MICs of amoxicillin and ticarcillin alone (>2,048 µg/ml) and combined with clavulanate (2,048 µg/ml), while MICs for the OXA-1-producing S. flexneri CFS02 were much lower (amoxicillin, 256 µg/ml; amoxicillin-clavulanate, 64 µg/ml; ticarcillin, 256 µg/ml; ticarcillin-clavulanate, 32 µg/ml). Against S. sonnei CFS01 (TEM-1 hyperproducing), clavulanate partially restored the activities of amoxicillin and ticarcillin (MICs, 64 µg/ml for both combinations).

No transconjugant was obtained by mating of S. sonnei UCK with Escherichia coli HB101 as recipient strain with ticarcillin (32 µg/ml) as selective agent. Plasmid DNA content was determined by comparison with plasmids Rsa (39 kb), TP114 (61 kb), pCFF04 (85 kb), and pCFF14 (180 kb). Strain UCK contained one large plasmid of ca. 90 kb which hybridized with the TEM probe (data not shown). Isoelectric focusing performed with polyacrylamide gels containing ampholines with a pH range of 3.5 to 10.0 showed that the -lactamase produced by S. sonnei UCK focused at a pI of 5.4.

The nucleotide sequence analysis obtained by direct sequencing of the PCR product revealed that the sequence of the bla gene differed from that of bla_TEM-1b at two positions: a G-162T transversion (at position 1 of the 10 consensus sequence) in the promoter region and the change A-407C, leading to the amino acid substitution Met-69Leu in the coding region. The latter change has already been observed in the inhibitor-resistant TEM -lactamase IRT-5/TEM-33 (4).

Table 1 shows the sequences of the structural genes and of the promoter regions specifying this IRT enzyme. Three different bla_TEM-33 genes which had the mutation A-407C in common were reported by Goussard and Courvalin (3): bla_TEM-33a, derived from bla_TEM-1b with the change T-226C, which is under the control of the weak P3 promoter (9); bla_TEM-33b, derived from bla_TEM-1b, which is under the control of the strong promoters Pa an Pb; and bla_TEM-33c, derived from bla_TEM-2 with the change A-317C (Lys-39Gln), with the promoter region corresponding to the strong P4 promoter with the T-32C amd G-162T mutations. The structural gene of IRT-5/TEM-33 produced by S. sonnei UCK is identical to bla_TEM-33b. The promoter region had the change G-162T (P4), which has been shown to be responsible for the hyperproduction of TEM-1 and which is commonly found upstream from the genes for inhibitor-resistant -lactamases (3). This fourth gene coding for TEM-33 was designated bla_TEM-33b-like.

View this table: [in this window] [in a new window]   TABLE 1.   Substitutions in blaTEM genes and derived genes encoding the inhibitor-resistant -lactamase TEM-33/IRT-5

Moderate resistance to amoxicillin-clavulanate (MIC₉₀ = 16 µg/ml) has been frequently reported in S. flexneri isolates, which often produce a relatively inhibitor-resistant OXA-type enzyme (2, 8).

The high-level resistance to amoxicillin-clavulanate (2,048 µg/ml) associated with susceptibility to cephalothin (8 µg/ml) observed in S. sonnei (UCK strain) suggested the presence of a hitherto unreported TEM-33 variant in the species. The sequence diversity observed for the IRT-3/TEM-33 genes lends weight to the idea that there is a great variety of TEM genes in nature, as previously suggested by Goussard and Courvalin (3) and as recently reported by Leflon-Guibout et al. (5). In this study, the patient had received no antibiotics before the resistant strain was isolated.