Early Bactericidal Activity of Paromomycin (Aminosidine) in Patients with Smear-Positive Pulmonary Tuberculosis

The sequence of the blaARI-1 gene from imipenem-resistant Acinetobacter baumannii 6B92 has been determined. The structural gene encodes a 273-amino-acid protein which is most related to the OXA class D b-lactamases. The conserved S-T-F-K and K-T-G motifs were identified in the ARI-1 protein sequence, also named OXA-23, but significantly, a point mutation (Y3F) was identified in the Y-G-N conserved motif, also known to function in the active site. Multiresistant Acinetobacter baumannii strains are now recognized as serious nosocomial pathogens (4, 5), and carbapenem-resistant strains are being reported increasingly (1, 3, 6, 11, 15, 18, 23). Imipenem-resistant A. baumannii 6B92 was isolated from a patient in Edinburgh, United Kingdom, in 1985 (15). Imipenem resistance was attributed to a novel serine b-lactamase, ARI-1 (15), and was subsequently demonstrated to be transferable to Acinetobacter junii (18). Imipenem resistance due to b-lactamases in A. baumannii has subsequently been reported worldwide, and two additional b-lactamases, ARI-2 (6) and an oxacillin-hydrolyzing enzyme (2, 11), defined only by their biochemical properties, have been described. In this paper we report the nucleotide and deduced amino acid sequences of ARI-1 carried by the R plasmid pUK1356. Bacterial strains and plasmids. The transconjugant A. junii BD413-2(pUK1356) was used as the source of ARI-1. Escherichia coli TG2 {supE hsdD5 thi D(lac-proAB) D(slr-recA) 306::Tn10(tet r )F 9[traD36 proAB 1 lacI q lacZDM15]} (17) was used as a host for recombinant plasmids prepared in the vector pUC19 (24). b-Lactamase purification and N-terminal amino acid sequencing. Cell extracts of A. junii BD413-2(pUK1356) were loaded onto a Mono Q anion-exchange column (Pharmacia Co. Ltd., Uppsala, Sweden) equilibrated with 50 mM Tris-HCl (pH 8.2). Fractions were eluted with a gradient of 0 to 0.5 M NaCl in 50 mM Tris-HCl (pH 8.2). The ARI-1 b-lactamase, eluted in the unadsorbed fraction, was loaded onto a Superdex-75 gel filtration column (Pharmacia) equilibrated in 50 mM Tris-HCl (pH 7.5) containing 0.1 M NaCl. Fractions containing b-lactamase activity, detected by an assay with nitrocefin (Glaxo Group Research Ltd., Greenford, United Kingdom), were concentrated by centrifugation using VectaSpin microtube filters, with a molecular mass cutoff of 12 kDa (Whatman International Ltd., Maidstone, United Kingdom). Following native PAGE, ARI-1 was transferred to a Problot membrane (PE Applied Biosystems, Warrington, United Kingdom), stained with Coomassie blue, and cut out for N-terminal sequencing with a model 477A gas phase sequencer (PE Applied Biosystems). The 20 N-terminal amino acids were used to prepare the degenerate oligonucleotide probe ARI-N (Table 1). Restriction enzymes and DNA cloning. Restriction enzymes (Life Technologies Ltd., Paisley, United Kingdom) were used in accordance with the manufacturer’s instructions. Plasmid pUK1356 was extracted, restricted, and cloned with the procedures described by Sambrook et al. (17). Recombinant clones were selected on nutrient agar (IDG Ltd., Bury, United Kingdom) containing X-Gal (5-bromo-4-chloro-3-indolyl-b-Dgalactopyranoside; 32.5 mg/liter), IPTG (isopropyl-b-D-thiogalactopyranoside; 7.8 mg/liter), and ampicillin (50 mg/liter; Sigma Aldrich Co. Ltd., Poole, United Kingdom). Hybridization and identification of the 5* end of the ARI-1 gene. Enhanced chemiluminescence probe labelling, hybridization, and detection kits (Amersham Pharmacia Biotech UK Ltd., Amersham, United Kingdom) were used in accordance with the manufacturer’s instructions. A partial library of pUK1356 recombinant clones was screened with an ARI-N probe 39 end labelled with fluorescein-dUTP. Recombinant plasmid from a single positive clone was purified with a Qiagen (Crawley, United Kingdom) plasmid minikit and sequenced with an ABI PRISM 377 automated DNA sequencer (PE Applied Biosystems). The sequence (Fig. 1, nucleotides 1 to 1345) revealed 375 bp of the 59 end of the blaARI-1 gene. PCR amplification and complete sequencing of the ARI-1 gene. PCRs were carried out in 100-ml volumes containing 16 mM (NH4)2SO4, 67 mM Tris-HCl (pH 8.8), 0.1% Tween 20 buffer, 2.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphates (Amersham Pharmacia Biotech UK Ltd.), 0.1 to 0.5 mM each primer, an d1Uo fBIOTAQ polymerase (Bioline UK Ltd., London, United Kingdom) or 1.25 U of Pfu DNA polymerase