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Antimicrobial Agents and Chemotherapy, February 2007, p. 631-637, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.01082-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Novel Ambler Class A ß-Lactamase LAP-1 and Its Association with the Plasmid-Mediated Quinolone Resistance Determinant QnrS1{triangledown}

Laurent Poirel,1 Vincent Cattoir,1,2 Ana Soares,1 Claude-James Soussy,2 and Patrice Nordmann1*

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris-Sud, K.-Bicêtre,1 Service de Bactériologie-Virologie, Hôpital Henri Mondor, Créteil, France2

Received 28 August 2006/ Returned for modification 8 October 2006/ Accepted 14 November 2006


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ABSTRACT
 
The plasmid-mediated quinolone resistance determinant QnrS1 was identified in non-clonally related Enterobacter cloacae isolates in association with a transferable narrow-spectrum ß-lactam resistance marker. Cloning experiments allowed the identification of a novel Ambler class A ß-lactamase, named LAP-1. It shares 62 and 61% amino acid identity with the most closely related ß-lactamases, TEM-1 and SHV-1, respectively. It has a narrow-spectrum hydrolysis of ß-lactams and is strongly inhibited by clavulanic acid and sulbactam and, to a lesser extent, by tazobactam. Association of the blaLAP-1 gene with the qnrS1 gene was identified in E. cloacae isolates from France and Vietnam. These genes were plasmid located and associated with similar insertion sequences but were not associated with sul1-type class 1 integrons, as opposed to the qnrA genes.


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INTRODUCTION
 
Whereas numerous ß-lactamases are increasingly reported worldwide, the diversity of acquired narrow-spectrum penicillinases remains quite limited in gram-negative organisms. They are mostly Ambler class A ß-lactamases of the TEM, SHV, or CARB types (1). Most of the TEM- and SHV-type enzymes are inhibited by clavulanic acid, and many variants have a hydrolysis spectrum expanded to cephalosporins. Their genes are mostly plasmid located.

Plasmid-mediated determinants conferring resistance to quinolones (QnrA, QnrB, and QnrS) are increasingly reported in Enterobacteriaceae (15). Those pentapeptide repeat proteins usually confer resistance to nalidixic acid and reduced susceptibility to fluoroquinolones (14, 23). QnrA1 and QnrS1 share 59% amino acid identity, whereas both determinants share 40% amino acid identity with QnrB1. Association of the QnrA determinant with clavulanic acid-inhibited extended-spectrum ß-lactamases (ESBL) (VEB-1, SHV-7, CTX-M-9, and CTX-M-14 [15, 22]) has been reported repeatedly. The qnrA-like genes have been identified in a large variety of enterobacterial species systematically associated with sul1-type integrons, involving the insertion sequence ISCR1 (formerly CR1 element) (27), likely at the origin of their acquisition and source of their expression (13, 24, 25, 28). The qnrB1 gene was first identified on a plasmid that carried the ESBL gene blaCTX-M-15 in a Klebsiella pneumoniae isolate from India and then in several isolates of Enterobacter cloacae, Citrobacter koseri, and Escherichia coli from the United States (8). Other QnrB variants have been subsequently identified, with QnrB2 and QnrB5 being identified in non-Typhi Salmonella enterica isolates from the United States (4). The qnrS1 gene was associated with the gene encoding the narrow-spectrum penicillinase TEM-1 in Shigella flexneri and E. cloacae from Asia (6, 21). The qnrS1 gene was also identified in Salmonella enterica serotype Bovismorbificans from the United States(4) and Salmonella enterica serotype Infantis from Germany (10), whereas the qnrS2 gene was found in S. enterica serotype Anatum from the United States (4).

Recently, by analyzing the prevalence of QnrA and QnrS resistance determinants in a collection of ESBL+ and ESBL– enterobacterial isolates recovered at the Bicêtre Hospital (France) from 2002 to 2005, the very first qnrS gene in Europe was identified (19). This qnrS1 gene was identified in six E. cloacae isolates corresponding to four distinct genotypes, a single E. coli isolate, and a single Serratia marcescens isolate. When transferring the plasmid harboring the QnrS1 determinant in an E. coli recipient strain, we had observed that an ampicillin resistance marker had been cotransferred that was not related to a known ß-lactamase (preliminary PCR analysis). Thus, the aim of the present study was to characterize this novel ß-lactamase and to identify the genetic structures linking ß-lactam and quinolone resistance determinants in these qnrS1-positive isolates.


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MATERIALS AND METHODS
 
Bacterial strains. Enterobacterial isolates were identified with the API20E system (bioMérieux, Marcy l'Etoile, France). The QnrS1-positive isolates included in the study were E. cloacae S1 to S6, E. coli S7, and S. marcescens S8 from our hospital (19) and also E. cloacae 287 from Vietnam (21). These isolates produced the same QnrS1 determinant. The QnrA-positive isolates were E. cloacae A1, Enterobacter aerogenes A2, and K. pneumoniae A3 (19). E. coli DH10B was the host for cloning experiments, and azide-resistant E. coli J53 was used as the recipient strain for conjugation and transformation experiments (13).

Susceptibility testing. Antibiotic-containing disks were used for routine antibiograms performed by disk diffusion testing (Sanofi-Diagnostic Pasteur, Marnes-la-Coquette, France), as previously described (9). MICs were determined by an agar dilution technique, as described previously (20). MICs of ß-lactams were determined alone or in combination with a fixed concentration of clavulanic acid (4 µg/ml) or tazobactam (4 µg/ml). MIC results were interpreted according to the guidelines of the CLSI (2).

PCR and hybridization experiments. Total DNA of enterobacterial isolates was extracted as described previously (25). This DNA was used as a template under standard PCR conditions (25) with a series of primers designed for the detection of class A ß-lactamase genes blaTEM, blaSHV, and blaCARB (5, 13, 18). Southern hybridizations were performed as described by Sambrook et al. (25) using the ECL nonradioactive labeling and detection kit (Amersham Pharmacia Biotech, Orsay, France). Screening of the blaLAP-1 gene among our strains was performed by PCR using primers LAP-1A (5'-CAATACAAAGCACAGAAGACC-3') and LAP-1B (5'-CCGATCCCTGCAATATGCTC-3') (Fig. 1), and this PCR product was used as a specific probe for detection of the blaLAP-1 gene.


Figure 1
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  		FIG. 1. Genetic environments of the blaLAP-1 and qnrS1 genes. Open reading frames are indicated by horizontal arrows. The right and left inverted repeats of Tn3 (IRR and IRL, respectively) are indicated. The restriction sites which have been used for cloning are indicated. Both inverted repeat extremities of the invertase element are shown. Plasmid pAH0376 is from S. flexneri from Japan (6), plasmid pINF5 is in S. enterica serotype Infantis (10), and plasmids pS3-1 and pS5-1 were obtained in this study. The IS26-related sequences identified only in S. enterica serotype Infantis are indicated. The structure identified by PCR mapping in isolates S4 and 287 is also indicated. The question mark indicates an unknown sequence. Nomenclature of the genes is according to that provided in reference 10. Positions of primers are indicated by arrows; primers 1, 2, 3, 4, 5, 6, 7, and 8 are primers PBP3B, LAP-1A, LAP-1B, orfB-B, 213A, 213B, qnrS-5'ext, and qnrS-3'ext, respectively.

Cloning experiments, recombinant plasmid analysis, DNA sequencing, and PCR mapping. Total DNAs of E. cloacae S3 and S5 isolates were restricted with EcoRI or with EcoRI and SacI, ligated into the corresponding sites of plasmid pBK-CMV, and then transformed into the E. coli TOP10 reference strain, as described elsewhere (16), giving rise to recombinant plasmids pS3-1 (containing an EcoRI fragment) and pS5-1 (containing an EcoRI-SacI fragment), respectively. Recombinant plasmids were selected onto trypticase soy agar plates containing amoxicillin (30 µg/ml) and kanamycin (30 µg/ml). The cloned DNA fragments were sequenced on both strands with an Applied Biosystems sequencer (ABI 3100; Foster City, Calif.). The nucleotide and deduced amino acid sequences were analyzed and compared to sequences available over the Internet at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov).

PCR mapping was performed to identify the genetic structures surrounding the qnrS1 gene in the different isolates tested. Primers specific for the blaLAP-1 gene were used in combination with primers PBP3B (5'-CGTCCTGAAGTGGCACTGG-3'), orfB-B (5'-CAGCAGTCCTGCGCGAAGG-3'), 213A (5'-GCTTCAGCCTCAGCGTCAAG-3'), 213B (5'-CGATGAATCAGGCTCCAGTC-3'), qnrS-5'ext (5'-GCGAATGAATGTGCAAGCGG-3'), and qnrS-3'ext (5'-GAACTCGACGGTTTAGATCC-3') (Fig. 1).

Genetic support. Mating-out assays were performed with the blaLAP-1-positive isolates as donors and azide-resistant E. coli J53 as recipient strain, as described previously (13). Plasmid DNAs of the qnrS1-positive isolates were extracted using the Kieser method (11).

ß-Lactamase purification and isoelectric focusing (IEF) analysis. Cultures of E. coli DH10B(pS3-1) were grown overnight at 37°C in 4 liters of trypticase soy broth containing amoxicillin (30 µg/ml) and kanamycin (30 µg/ml). ß-Lactamase was purified by ion-exchange chromatography. Briefly, the ß-lactamase extract was sonicated, cleared by ultracentrifugation, treated with DNase, and dialyzed against 20 mM bis-Tris buffer (pH 6.1). This extract was loaded on a Q-Sepharose column equilibrated with the same buffer, and the ß-lactamase-containing fractions were eluted in the flowthrough. After dialysis against 20 mM bis-Tris buffer (pH 8), the ß-lactamase was reloaded on the Q-Sepharose column equilibrated with the same buffer, and the ß-lactamase activity was retained in the column and then eluted using a NaCl gradient. Finally, the fractions containing the highest ß-lactamase activity were pooled and concentrated using an ultrafiltration filter tip (Sartorius, Goettingen, Germany). The purity of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (25).

IEF analysis was performed with an ampholine polyacrylamide gel (pH 3.5 to 9.5), as described previously (16), using purified ß-lactamase extracts from a culture of E. coli DH10B(pS3-1). The focused ß-lactamases were detected by overlaying the gel with 1 mM nitrocefin (Oxoid, Dardilly, France) in 100 mM phosphate buffer (pH 7.0).

Kinetic measurements. Purified ß-lactamase was used for kinetic measurements performed at 30°C with 100 mM sodium phosphate (pH 7.0) with an ULTROSPEC 2000 UV spectrophotometer (Amersham Pharmacia Biotech) (17). The 50% inhibitory concentrations (IC50s) were determined for clavulanic acid, tazobactam, sulbactam, cefoxitin, and imipenem. Various concentrations of inhibitors were preincubated with the purified enzyme for 3 min at 30°C to determine the concentrations that reduced the hydrolysis rate of 100 µM benzylpenicillin by 50%.

The specific activity of the purified ß-lactamase from E. coli DH10B(pS3-1) was obtained as described previously (16). One unit of enzyme activity was defined as the activity which hydrolyzed 1 µmol of benzylpenicillin per min per mg of protein. The total protein content was measured with the DC protein assay kit (Bio-Rad, Ivry-sur-Seine, France).

Nucleotide sequence accession number. The nucleotide sequence data reported in this work have been deposited in the GenBank nucleotide database under accession no. EF026092.


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RESULTS
 
Cloning and sequencing of the ß-lactamase gene. Preliminary attempts to detect by PCR several Ambler class A ß-lactamase-encoding genes failed (data not shown). Using total DNA of E. cloacae S3 as a template in cloning experiments, the pS3-1 recombinant plasmid was obtained. Sequence analysis of the 12-kb cloned EcoRI fragment of pS3-1 revealed an 858-bp-long open reading frame (ORF) encoding a 285-amino-acid preprotein. This protein was a ß-lactamase designated LAP-1 and possessed the STFK, SDN, and KTG structural elements characteristic of the active site of Ambler class A ß-lactamases (Fig. 2) (1). The G+C content of blaLAP-1 was 43.8%. ß-Lactamase LAP-1 was distantly related to other class A ß-lactamases. The highest percentages of amino acid identity were 62% and 61% with TEM-1 and SHV-1, respectively, whereas LAP-1 shared only 43% identity with CARB-2 (PSE-1) (Fig. 3).


Figure 2
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  		FIG. 2. Comparison of the amino acid sequence of ß-lactamase LAP-1 to those of ß-lactamases TEM-1 and SHV-1. Dashes indicate identical amino acid residues. Critical residues which are involved in the extension of the substrate profile in TEM or SHV enzymes and that are conserved in LAP-1 with respect to TEM-1 or SHV-1 are shaded in gray. Boldfaced amino acids are residues in conserved motifs. Numbering is according to the method of Ambler (1).


Figure 3
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  		FIG. 3. Dendrogram obtained for representative Ambler class A ß-lactamases by neighbor-joining analysis. The alignment used for the tree calculation was performed with ClustalX. Branch lengths are drawn to scale and are proportional to the number of amino acid changes. The distance along the vertical axis has no significance. The scale bar represents a 10% difference in amino acid sequence. Only the bootstrap values superior to 85% are indicated. The amino acid identities of each ß-lactamase compared to LAP-1 are indicated in square brackets. Acquired narrow-spectrum ß-lactamases in gram-negative organisms are ROB-1, CARB-2, LAP-1, TEM-1, and SHV-1.

Distribution of the blaLAP-1 gene. Since the blaLAP-1 gene was identified in a qnrS-positive isolate, the prevalence of this ß-lactamase gene was evaluated in a collection of qnrS-positive isolates by PCR amplification. Eight QnrS-positive isolates recovered in our hospital during the 2005-2006 period were tested, including six E. cloacae isolates (including isolates S1 and S2, producing ESBLs SHV-12 and TEM-52, respectively), one E. coli isolate producing the ESBL CTX-M-1, and one TEM-1-positive S. marcescens isolate. PCR results indicated that five E. cloacae isolates (S1, S3, S4, S5, and S6) were positive for blaLAP-1. Pulsed-field gel electrophoresis analysis showed that these five E. cloacae isolates corresponded to three distinct genotypes, isolates S1 and S4 on one hand and isolates S5 and S6 on the other hand being clonally related (data not shown). In addition, the QnrS1-positive E. cloacae isolate 287 from Vietnam (21) was also found to harbor the blaLAP-1 gene.

None of the 10 QnrA1-positive enterobacterial isolates recovered from our hospital or from other countries such as Canada, Australia, and Thailand was positive for the blaLAP-1 gene. In addition, since the five blaLAP-1-positive isolates were E. cloacae, we searched for the blaLAP-1 gene among a collection of 30 randomly selected nonreplicate E. cloacae isolates recovered in our hospital in 2006. PCR revealed that these isolates were all negative for the blaLAP-1 gene.

Antibiotic susceptibility. E. cloacae S3 was resistant to kanamycin and tobramycin (MICs of >256 µg/ml), tetracycline, rifampin, and fluoroquinolones (MICs of ofloxacin and ciprofloxacin of >32 µg/ml) (19). MICs of ß-lactams for E. cloacae S3 and for E. coli DH10B(pS3-1) indicated the expression of a clavulanic acid-inhibited ß-lactamase that spared expanded-spectrum cephalosporins and carbapenems (Table 1). MICs of ß-lactams for other blaLAP-1-positive isolates were distributed in the same ranges (data not shown).


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  		TABLE 1. MICs of ß-lactams for E. cloacae clinical isolate S3, E. coli DH10B harboring recombinant plasmid pS3-1 expressing ß-lactamase LAP-1, and the E. coli DH10B reference strain

Biochemical properties of LAP-1. IEF analysis showed that E. coli DH10B(pS3-1) had a ß-lactamase activity with a pI value of 6.7 (data not shown). ß-Lactamase activity with a pI value of 8.5 was also detected for E. cloacae S3, corresponding to the chromosomal cephalosporinase of E. cloacae. The specific activity of the purified ß-lactamase LAP-1 was 350 U·mg of protein–1. Its overall recovery was 70% with a 90-fold purification. The purity of the enzyme was estimated to be more than 95% according to sodium dodecyl sulfate-gel electrophoresis analysis (data not shown).

Kinetic parameters of LAP-1 showed its narrow-spectrum activity against ß-lactams, including penicillins and cephalothin but excluding most expanded-spectrum cephalosporins (except cefepime), cephamycins, and carbapenems (Table 2). IC50 determinations performed with benzylpenicillin as substrate showed that LAP-1 activity was inhibited by clavulanic acid (IC50, 0.08 µM) and sulbactam (IC50, 0.2 µM), and to a lesser extent by tazobactam (IC50, 10 µM). In addition, LAP-1 activity was inhibited by cefoxitin at a lower level (IC50, 100 µM) but was not inhibited by imipenem.


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  		TABLE 2. Kinetic parameters of purified ß-lactamase LAP-1a

Genetic environment of blaLAP-1. Sequencing of the 12,935-bp insert fragment of plasmid pS3-1 from E. cloacae S3 revealed several ORFs. Upstream of blaLAP-1, an ORF was present that encoded a protein sharing 53 and 52% amino acid identity with a transpeptidase encoded by the genome of Salmonella enterica subsp. enterica and of E. coli, respectively (GenBank numbers YP151544 and ZP009224346). The ORF ends 64 bp upstream of the start codon of the blaLAP-1 gene and was truncated in correspondence to the EcoRI site used for cloning. Downstream of the blaLAP-1 gene, the qnrS1 gene was identified (Fig. 1). The two resistance genes were separated by 1,597 bp that included the insertion sequence ISEcl2, previously identified upstream of the qnrS1 gene in E. cloacae 287 (21) and located 11 bp downstream of blaLAP-1. ISEcl2 is a 1,282-bp insertion sequence belonging to the IS3 family and possessing 24-bp-long inverted repeats. It was made of two ORFs, OrfA being likely truncated by a single base substitution and encoding a 53-amino-acid-long protein and OrfB, encoding a 218-amino-acid-long protein (Fig. 1). No target site duplication was observed at the ISEcl2 ends. In silico analysis showed that this ISEcl2 element was also present in the qnrS1-positive pINF5 plasmid of S. enterica serotype Infantis (with a few nucleotide substitutions that led to a longer OrfA product of 122 amino acids) (10), where it has been truncated at its 3'-end extremity by the insertion of Tn3 (Fig. 1). Downstream of the qnrS1 gene, the same features identified in S. enterica serotype Infantis were found, including a truncated resolvase protein and a segment showing significant homology with the E. coli CS12 fimbrial gene cluster. This region of homology corresponded likely to an inversion system. This element was 2,850-bp long, possessed 14-bp inverted repeats, and contained four open reading frames, one sharing a significant degree of homology with the resolvase of ISXc5, a peculiar transposable element (12). A switch between the sequence from Salmonella and that identified in pS3-1 was observed exactly after a TTATT site, which was possibly the original target site of a blaTEM-1-borne Tn3 insertion in S. enterica serotype Infantis and was also present at the right-hand end of the invertase element (Fig. 1) (10). At the right extremity of that site, no IS26-related features were identified, by contrast to the sequence of S. enterica serotype Infantis (10). Sequence analysis revealed an open reading frame sharing 53% amino acid identity with the FipA protein of plasmid pKM101 of E. coli (26) or plasmid pGFT1 of Salmonella enterica serovar Dublin (3), these proteins being known to be expressed by conjugative broad-host-range plasmids, such as some IncN plasmids, and to represent a fertility inhibition protein that inhibits conjugal transfer of cohabitating IncP plasmids.

Interestingly, detailed analysis of recombinant plasmid pS5-1, obtained from E. cloacae isolate S5 and harboring blaLAP-1 and qnrS1 genes, showed that its sequence was very similar to that observed in plasmid pS3-1, except that the invertase element, located exactly at the same position, was in the opposite orientation (Fig. 1). However, at the right-hand extremity of that element, no TTATT site was identified and the sequence differed from that of pS3-1, with the presence of an ORF encoding a 134-amino-acid protein of unknown function.

Subsequent PCR mapping revealed that the structures surrounding the blaLAP-1 and qnrS1 genes in E. cloacae S1 and S6 were identical to those identified in E. cloacae S5 (data not shown). However, in isolates S4 and 287, PCR mapping revealed that the invertase element was absent. In these two latter isolates, the sequences present upstream of the blaLAP-1 gene were identical to those identified in isolates S3 and S5, whereas those present downstream of the blaLAP-1 gene differed (Fig. 1).

Genetic support of the ß-lactamase determinant. Conjugation experiments allowed the transfer of a ca. 100-kb plasmid encoding ß-lactamase LAP-1 from E. cloacae S3 and S4 isolates to recipient strain E. coli J53. These transconjugants exhibited an identical narrow-spectrum resistance profile to ß-lactams, including resistance to ticarcillin antagonized by clavulanic acid addition and also a reduced susceptibility to piperacillin (Table 1). In addition, these transconjugants were resistant to nalidixic acid and had reduced susceptibility to fluoroquinolones, whereas no additional antibiotic resistance marker was cotransferred. Using the different blaLAP-1-positive isolates as donors, conjugation experiments allowed us to obtain blaLAP-1-positive transconjugants from E. cloacae S3 and S4 isolates but not with the other isolates as donors. However, plasmid analysis showed that the five qnrS-positive isolates possessed a similar-in-size plasmid of ca. 100 kb that cohybridized with the blaLAP-1- and the qnrS-specific probes (data not shown). The plasmid coharboring the blaLAP-1 and the qnrS1 genes in E. cloacae 287 was 50 kb in size (19).


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DISCUSSION
 
This study identified a totally novel class A ß-lactamase that had weak amino acid identity with known ß-lactamases. The ß-lactamase LAP-1 is one of the very few acquired narrow-spectrum class A ß-lactamases described so far in gram-negative organisms. Most of the acquired ß-lactamases reported recently in gram negatives have an extended-spectrum resistance profile, such as CTX-M, PER, VEB, BEL, BES, KPC, and IMI enzymes (7). Detailed analysis of its amino acid sequence showed that LAP-1 shared with the narrow-spectrum ß-lactamases TEM-1 or SHV-1 the same amino acids, such as Asp104, Gly156, Arg164, Leu169, Asp179, and Gly238 (Fig. 2). Amino acid substitutions at those positions have been reported to be the source of extension of the resistance profiles (http://www.lahay.org/studies/). This observation correlates well with the narrow-spectrum resistance profile of LAP-1.

Analysis of the surrounding sequences of the blaLAP-1 gene showed that it was located next to the plasmid-encoded quinolone resistance gene qnrS1, those two genes being probably part of the same mobilizable unit. Interestingly, the GC content of the qnrS1 gene (43.8%) is exactly identical to that of the blaLAP-1 gene. This might indicate that both antibiotic resistance genes come from the same ancestor. The blaLAP-1 gene is not part of a gene cassette in a class 1 integron structure and is not associated with any ISCR element (27, 28), as opposed to what is known now for recently identified ß-lactamase genes, such as the blaVEB, blaGES, blaBEL, and blaCTX-M genes.

It is quite surprising that the blaLAP-1 gene has disseminated in non-clonally related E. cloacae isolates from different parts of the world, although its occurrence in the S. flexneri isolate from Japan, the S.enterica serotype Infantis isolate from Germany, and the Salmonella isolates from the United States is yet unknown. This association between the blaLAP-1 gene and the qnrS1 gene might provide resistance or reduced susceptibility to both narrow-spectrum ß-lactams and quinolones. This association may be quite frequent, and further extended surveys might analyze the distribution of this novel ß-lactamase gene in Enterobacteriaceae to evaluate whether it represents an emerging threat.


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ACKNOWLEDGMENTS
 
This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, France, and mostly by grants from the European Community (6th PCRD, LSHM-CT-2003-503335, and LSHM-CT-2005-018705). L.P. is a researcher from the INSERM (Paris, France). We thank L. Peixe for support of the work of A.S.

We thank T. Naas for fruitful discussions.


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FOOTNOTES
 
* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice{at}bct.aphp.fr. Back

{triangledown} Published ahead of print on 20 November 2006. Back


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Antimicrobial Agents and Chemotherapy, February 2007, p. 631-637, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.01082-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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