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Antimicrobial Agents and Chemotherapy, February 2006, p. 781-784, Vol. 50, No. 2
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.2.781-784.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

New TEM-Derived Extended-Spectrum ß-Lactamase and Its Genomic Context in Plasmids from Salmonella enterica Serovar Derby Isolates from Uruguay

Rafael Vignoli,1 Nicolas F. Cordeiro,1 Virginia García,1 María Inés Mota,1 Laura Betancor,2 Pablo Power,3 José A. Chabalgoity,2 Felipe Schelotto,1 Gabriel Gutkind,3 and Juan A. Ayala4*

Departamento de Bacteriología y Virología,1 Departamento de Biotecnología, Instituto de Higiene, Facultad de Medicina, Universidad de la República, 11600 Montevideo, Uruguay,2 Cátedra de Microbiología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Buenos Aires, Argentina,3 Centro de Biología Molecular "Severo Ochoa," CSIC-UAM, Campus de Cantoblanco, 28049 Madrid, Spain4

Received 29 July 2005/ Returned for modification 1 September 2005/ Accepted 4 November 2005


   ABSTRACT
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A small (8.2-kb) ColE1 plasmid encoding TEM-144 (a new ß-lactamase with a ceftazidimase profile) was sequenced by a gene-walking strategy. The blaTEM allele was carried on a Tn2 element, disrupting a Rom protein gene. TEM-144 differs from TEM-1 by two mutations (R164C and E240K) and from the ceftazidime-hydrolyzing TEM-91 by one mutation (T182M).


   TEXT
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The TEM-derived extended-spectrum ß-lactamases (ESBLs) include more than 100 variants (11) originated by one to four amino acidic modifications relative to TEM-1 or TEM-2. Modifications of a few residues have been described to be responsible for TEM-derived ESBL activity, namely, Glu-104, Arg-164, Ala-237, Gly-238, and Glu-240 (17). Interestingly, blaTEM genes are frequently embedded in transposable elements such as Tn2 inserted in wide-host-range plasmids (12).

Two Salmonella enterica serovar Derby isolates with decreased susceptibility to ceftazidime were detected in a previous study of samples of poultry origin (6). An 8.2 kb-plasmid (pSD1 and pST12) was obtained from isolates SD1 and ST12. Both plasmids showed identical restriction patterns when digested with PstI, displaying three fragments of about 5, 3, and 0.7 kb (data not shown), and could be transferred by transformation (24) to Escherichia coli strain CAG12177 [F– {lambda}– zej-298::Tn10(Tetr) gyrA261(Nalr) rph-1] (E. coli Genetic Stock Center). MICs were determined as specified by the CLSI (formerly NCCLS) (18). Both transformants CAG12177/pSD1 and CAG12177/pST12 had identical antibiotic susceptibility profiles, which were also similar to those of isolates SD1 and ST12 (Table 1). The MICs of ceftriaxone and ceftazidime, with and without clavulanic acid, suggested that they produced enzymes hydrolyzing ceftazidime more efficiently than ceftriaxone (Table 1). Transformants remained fully susceptible to the carbapenems and to other families of antibiotics such as aminoglycosides, tetracyclines, quinolones, and sulfonamides.


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  		TABLE 1. Antibiotic susceptibility of S. enterica serovar Derby SD1 and ST12 isolates and transformants

 
Crude extracts of both original and transformant strains displayed, after isoelectrofocusing (21), a single ß-lactam-hydrolyzing band at an apparent pI of 5.6 when revealed with 0.5 mM nitrocefin (data not shown).

PCRs (25) with specific blaTEM primers were positive when plasmid DNA (both from the original poultry isolates as well as from the transformants) was used as a template but yielded negative results with primers specific for blaSHV, blaCTX-M, blaPER-2, and blaOXA genes (for primer descriptions, see Table 2). Sequence analysis of PCR products revealed the presence of a blaTEM allele encoding a new TEM-derived enzyme, showing two amino acid differences compared to TEM-1 (R164C and E240K). These modifications occurred by two nucleotide substitutions (in boldface type): CGT->TGT at position 692 and GAG->AAG at position 917, respectively, as described previously by Ambler et al. (1). Only one difference could be detected compared with TEM-91 (13), namely, T182M (ATG->ACG). The amino acidic sequence of mature TEM-144 had a predicted pI of 5.6, consistent with the value previously observed by isoelectric focusing.


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  		TABLE 2. Primers used for PCR and sequencing

 
TEM-144 has two mutations known to be involved in the extension of substrate specificity of TEM-type enzymes and could explain the resistance profile conferred by this enzyme. A substitution at position 164 (Arg->Cys) could break a salt bridge formed between residues 164 and 179 in the {Omega}-loop, allowing the entrance of ceftazidime molecules in the oxyanionic pocket, as previously revealed by Majiduddin and Palzkill (16). It should be noted that of 40 TEM-derived ESBLs with R164 modifications, only two (TEM-87 [20] and TEM-91 [13]) contain an R164C substitution (11). On the other hand, substitutions of E240 were always E240K (11). However, only the following three TEM-derived ESBLs have only two modifications, including one at position 240 and another additional change: TEM-10 (R164S) (22), TEM-28 (R164H) (7), and TEM-71 (G238S) (23).

The low-level resistance to oxyimino cephalosporins except ceftazidime (Table 1) could hamper laboratory detection of this ESBL if the latter drug is not used in detection tests.

Eleven primers were designed during a gene-walking strategy (Table 2) to determine the full sequence of the 8.2-kb plasmids. Sequences were assembled using NCBI tools, resulting in a complete circular sequence (Fig. 1), and were identical for the two plasmids. The sequence contains three different regions encoding antimicrobial resistance, replication, and mobilization functions, respectively.


Figure 1
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  		FIG. 1. Circular genetic map of pST12. The inner circle represents the three zones with a known function. The truncated Rom protein gene is depicted in black. ORFs are indicated by arrows pointing in the direction of transcription. Gene names and their coordinates are displayed in boldface type. Restriction endonucleases and their cutting sites are shown in regular type. IRR, inverted repeat region.

 
The antimicrobial resistance region zone includes three open reading frames (ORFs) corresponding to the tnpA, tnpR, and blaTEM coding sequences. The blaTEM promoter was recognized upstream of blaTEM (regions –35TTCAAA and –10GACAAT) and belongs to the weak promoter type P3 with the following three conserved nucleotides: C32, G162, and G175 (14). This antibiotic resistance gene was incorporated into a transposable element identical to Tn2*, which was described previously by Partridge and Hall (19). A replicative region involved in the regulation of replication of ColE1-like plasmids (9) was found upstream of Tn2*. This region encodes RNA II (promotes plasmid replication) and RNA I (antisense down-regulation element) and contains a putative oriV. A fourth element, the gene rom (the only coding sequence in the replication origin), was truncated by Tn2 insertion (see below). Downstream of Tn2*, a horizontal plasmid mobilization system was detected, and this transmissibility region contains a mobA gene and oriT elements (5). An additional region is a multimer resolution site, an important mechanism for the regeneration of monomers in high-copy-number plasmids. The rest of the plasmid included four ORFs without any known function.

After excluding the Tn2 region from the plasmid, the queries for alignment yielded only three significant matches corresponding to three closely related plasmids, namely, pOSAK1 (gi4589704), p4821 (gi3152962) (10), and NTP16 (gi9507429) (8). pOSAK1 and p4821 are small plasmids without transposable elements, but NTP16 presents a complete Tn4352 that confers kanamycin resistance and a truncated Tn2 that confers ampicillin resistance. Interestingly, in pST12/pSD1 and in NTP16, a Tn2 element is inserted, interrupting a Rom protein gene, including the same "TTCTT" target sequence (8). The presence of Tn2 disrupting the gene rom could favor a higher plasmid copy number by inactivating the replication inhibitor (3). This fact may lead to at least two linked consequences: an increase in the copy number of blaTEM and consequently a larger amount of enzyme, which might have an impact on the level of resistance (15). Likewise, a larger number of blaTEM copies per bacterial cell could increase the chances of generating new variants of ß-lactamases with better hydrolytic capabilities.

Even if Salmonella enterica serovar Derby is not one of the serovars most frequently associated with human diarrhea, the fact that SD1 and ST12 were detected in the human food chain (hen eggs) points to the possibility of accession to the human digestive tract and spreading in the clinical setting.

Nucleotide sequence accession numbers. The sequences of TEM-derived ESBLs were deposited into the EMBL database under accession numbers AM049399 and AM049400.


   ACKNOWLEDGMENTS
 
We are grateful to Ana Castro and Elba Hernandez for technical assistance.

This work was partially supported by Fundación Manuel Pérez, Uruguay, and Comisión Sectorial de Investigación Científica (CSIC), Universidad de la República, Uruguay. Part of this work was also supported by grants from the LSHM-CT-2003-503335 European project to J.A.A. and a UBACYT, ANPCYT, and Carrillo-Oñativia fellowship to G.G.


   FOOTNOTES
 
* Corresponding author. Mailing address: Centro de Biología Molecular "Severo Ochoa," CSIC-UAM, Campus de Cantoblanco, 28049 Madrid, Spain. Phone: 34-91-4978083. Fax: 34-91-4978087. E-mail: jayala{at}cbm.uam.es. Back


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Antimicrobial Agents and Chemotherapy, February 2006, p. 781-784, Vol. 50, No. 2
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.2.781-784.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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