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Antimicrobial Agents and Chemotherapy, June 2004, p. 2321-2324, Vol. 48, No. 6
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.6.2321-2324.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of a Carbapenem-Hydrolyzing Class A ß-Lactamase, SFC-1, from Serratia fonticola UTAD54

Isabel Henriques,1 Alexandra Moura,1 Artur Alves,1 Maria José Saavedra,2 and António Correia1*

Center for Cell Biology, Department of Biology, University of Aveiro, 3810-193 Aveiro,1 CECAV, Department of Veterinary Science, University of Trás-os-Montes e Alto Douro, Vila Real, Portugal2

Received 11 September 2003/ Returned for modification 20 November 2003/ Accepted 6 February 2004


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ABSTRACT
 
An environmental isolate of Serratia fonticola resistant to carbapenems contains a gene encoding a class A ß-lactamase with carbapenemase activity. The enzyme was designated SFC-1. The blaSFC-I gene is contained in the chromosome of S. fonticola UTAD54 and is absent from other S. fonticola strains.


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TEXT
 
The prokaryotic species Serratia fonticola, a species of the family Enterobacteriaceae, includes organisms that occur naturally in environmental waters (3); occasionally, some strains cause infections in humans (15). A recent study on the natural antimicrobial susceptibilities of strains of Serratia species (23) showed that S. fonticola expresses both a chromosomally encoded extended-spectrum class A ß-lactamase and a species-specific AmpC ß-lactamase. The class A enzyme corresponds to the previously characterized ß-lactamase SFO-1 (13), and the homologous sequence FON-A (GenBank accession no. AJ251239) is common to S. fonticola (19).

In a previous report (18), an environmental isolate designated S. fonticola UTAD54 was shown to be resistant to carbapenems. This phenotype could be attributed to a gene encoding a class B metallo-enzyme (Sfh-I) that was isolated from a genomic library (18). An additional screening of the library was done on Luria-Bertani plates supplemented with ampicillin (50 µg/ml) and kanamycin (30 µg/ml) to select for inserts and the vector, respectively. Some of the clones obtained were negative when screened by PCR using primers (18) for genes homologous to SFO-1. A recombinant plasmid containing a 1.8-kb insert was selected for study and designated pIH18.

Characterization of a new ß-lactamase gene. Plasmid DNA was prepared with a Qiaprep kit (Qiagen, Courtaboeuf, France), and both strands of the insert were sequenced on an ABI cycle sequencer A373 (Applied Biosystems/Perkin-Elmer, Foster City, Calif.) using the ABI Prism dye terminator kit.

Analysis of sequence data revealed the presence of an open reading frame of 927 bp encoding a 33.6-kDa protein containing 309 amino acids (Fig. 1). Four nucleotides upstream of the ATG codon have the sequence AAGG, a putative ribosome-binding site (RBS). A typical –10 region (TATACT) was identified upstream from the RBS; no conserved –35 region could be assigned. Downstream, the open reading frame is a palindromic sequence (Fig. 1) which might form a hairpin loop in the mRNA, typical of a transcription terminator. The overall G+C content of blaSFC-1 (45.3%) is characteristic of genes of Enterobacteriaceae.



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  		FIG. 1. Nucleotide and deduced amino acid sequences of the SFC-1 gene and its upstream and downstream regions. The putative –10 region and a potential RBS are in bold. The inverted repeat sequences that can act as a terminator of transcription are shaded. The putative signal peptide for protein secretion is underlined. Amino acids that correspond to conserved domains of class A ß-lactamases are shown in bold.

A similarity search was performed with BLAST (1). SFC-1 had the highest similarity to the class A carbapenemases, in particular KPC-1 (62% identical) from Klebsiella pneumoniae (21), Sme-1 (58%), NMC-A (59%), and IMI-1 (59%) (12). Lower similarity scores were returned for the other class A ß-lactamases. No putative LysR-type regulator gene was identified upstream of the blaSFC-I gene, whereas such regulators are transcribed upstream of the genes coding for NMC-A, Sme-1, and IMI-1 (7, 8).

The software SignalP (11) identified a bacterial signal peptide of 26 amino acids in the amino-terminal sequence (Fig. 1). Cleavage of this signal peptide would yield a mature protein of 30.7 kDa with a pI of 7.95.

Within the mature protein, a serine-serine-phenylalanine-lysine tetrad (S-S-F-K) was found, as was a lysine-threonine-glycine (KTG) motif. These motifs (SXXK and KTG) are characteristic of serine ß-lactamases (16, 22). The nine invariant residues typical of class A enzymes (G45, S70, K73, P107, S130, D131, A134, E166, and G236) are conserved in the SFC-1 sequence. From the residues suggested to be important for class A carbapenemase activity (C69, S70, K73, H105, S130, R164, E166, N170, D179, R220, K234, S237, and C238), only S237 was not conserved in the SFC-1 sequence.

The deduced amino acid sequence of SFC-1 was aligned to the sequences of 15 class A ß-lactamases, using CLUSTAL W at the European Molecular Biology Laboratory website (http://www.embl-heidelberg.de/). The enzymes and their GenBank accession numbers were the following: KPC-1 (24) from K. pneumoniae (AAG13410), IMI-1 (17) from Enterobacter cloacae (AAR93461), Sme-1 (9) from Serratia marcescens (CAA82281), OXY-1 (2) from Klebsiella oxytoca (P22391), CITDI (14) from Citrobacter diversus (S19006), YENT (20) from Yersinia enterocolitica (Q01166), CTX-M-12 (19) from K. pneumoniae (AAG34108), CTX-M-14 (19) from Escherichia coli (CAC95170), Toho-1 (6) from E. coli (BAA07082), SFO-1 (6) from E. cloacae (BAA76882), FONA-3 from S. fonticola (CAB61639), SER_FON (13) from S. fonticola (P80545), TEM-1 (24) from E. coli (AAR25033), SHV-1 (24) from E. coli (P14557), and CARB-3 (4) from Pseudomonas aeruginosa (P37322). The dendrogram shown in Fig. 2 was derived from the alignment: SFC-1 clusters to the class A carbapenemases and is more closely related to a subgroup that includes the enterobacterial enzymes of extended hydrolytic spectrum.



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  		FIG. 2. Dendrogram obtained from the multiple sequence alignment of 15 class A ß-lactamases. The percent identity between the amino acid sequence of each enzyme and that of SFC-1 is indicated in brackets.

Susceptibility to antibiotics. The MICs were determined by the E-test method (Biodisk, Solna, Sweden), and susceptibility categories were allocated according to those described in reference 10. Table 1 shows the MICs for S. fonticola UTAD54, E. coli transformed with plasmid pIH18, and untransformed E. coli. The DNA insert encoding SFC-1 when replicating in E. coli confers resistance to ampicillin, amoxicillin, piperacillin, cephalothin, and aztreonam and reduced susceptibility to meropenem and imipenem, and its activity is inhibited by the class A ß-lactamase inhibitors. Such a resistance pattern is characteristic of a carbapenem-hydrolyzing class A ß-lactamase.


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  		TABLE 1. MICs of antibiotics for S. fonticola UTAD54, E. coli XL2 Blue(pIH18), and E. coli XL2 Blue (reference strain)

Chromosomal location of class A ß-lactamases in S. fonticola UTAD54. DNA from S. fonticola UTAD54 embedded in agarose was digested with I-CeuI (New England Biolabs, Hertfordshire, United Kingdom), and the resulting fragments were separated on a CHEF-DRII apparatus (Bio-Rad, Richmond, Calif.) (5). Six fragments were generated. After immobilization on nylon membranes, the I-CeuI-generated fragments were hybridized with three different probes: an rRNA gene probe, a probe specific to the naturally occurring class A ß-lactamase (SFO-1), and a blaSFC-I probe.

The probes were generated by PCR amplification in the presence of digoxigenin (Roche Molecular Biochemicals, Indianapolis, Ind.). For rRNA genes and the SFO-1 gene, the primers were previously reported (18). Specific primers were designed to amplify the blaSFC-I gene, SfcF (5'-GATCTCGAGAATGTCACGCACCGGTCGACTG-3'), and SfcR (5'-GATGAATTCTTAGAAGCCGATAGACTTTCC-3'). The probes for the SFC-1 and SFO-1 genes revealed two different I-CeuI bands, as shown in lanes 1 and 2 of Fig. 3; the probe for rRNA genes hybridized to the six I-CeuI bands (lane 3). These results thus indicate that both ß-lactamase genes are chromosomally encoded and apart from each other. Hybridization of the probe for blaSFC-I with DNA from S. fonticola strains LMG 7882T, DSM 9663, and CIP 103850 did not detect homologous sequences in these genomes.



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  		FIG. 3. Hybridizations to I-CeuI fragments generated from the genome of S. fonticola UTAD54 and separated by pulsed-field gel electrophoresis. Lane 1, hybridization with SFC-1 probe; lane 2, hybridization with probe for naturally occurring class A ß-lactamases of S. fonticola; lane 3, hybridization using a probe for rRNA genes; lane 4, concatemers of phage lambda DNA.

Concluding remarks. S. fonticola UTAD54 is an exceptional strain, carrying the naturally occurring ß-lactamases of S. fonticola and different classes of carbapenemases, SFC-1 and the previously reported metallo-enzyme Sfh-I. Those enzymes are not present in other S. fonticola strains. These exceptional characteristics could be the result of the acquisition of a genetic element by horizontal gene transfer.

Nucleotide sequence accession number. The nucleotide sequence reported here was deposited in GenBank under accession number AY354402.


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ACKNOWLEDGMENTS
 
This work was financed by the Fundação para a Ciência e a Tecnologia through grants to Artur Alves (SFRH/BD/10389/2002) and Isabel Henriques (SFRH/BD/5275/2001).


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FOOTNOTES
 
* Corresponding author. Mailing address: Center for Cell Biology, Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal. Phone: 351-234370778. Fax: 351-234426408. E-mail: acorreia{at}bio.ua.pt. Back


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Antimicrobial Agents and Chemotherapy, June 2004, p. 2321-2324, Vol. 48, No. 6
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.6.2321-2324.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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