A clinical isolate of Serratia marcescens (TN9106) produced a metallo 13-lactamase (IMP-1) which conferred resistance to imipenem and broad-spectrum 13-lactams. The consensus amino acid residues, His-95, His-97, Cys-176, and His-215, which form putative zinc ligands, were conserved in the deduced amino acid sequence of IMP-1. By determination of the amino acid sequence at the N terminus of purified mature IMP-1, 18 amino acid residues were found to be processed from the N terminus of the premature enzyme as a signal peptide. These results clearly show that IMP-1 is an enterobacterial metallo 13-lactamase, of which the primary structure has been completely determined, that confers resistance to carbapenems and other broad-spectrum 13-lactams.Many extended-spectrum 13-lactamases conferring high levels of resistance to broad-spectrum P-lactam antibiotics have been found worldwide with the increasing use of newly developed broad-spectrum P-lactam antibiotics (10, 11).They are mostly R plasmid-mediated TEM-or SHV-related class A enzymes (4,12,18,22,29,32). However, plasmidmediated AmpC-type ,-lactamases that belong to class C have recently been reported (9, 21). These plasmid-mediated extended-spectrum ,B-lactamases have a serine residue at the active center of the enzyme, and they effectively hydrolyze broad-spectrum ,-lactams, except carbapenems. Although it was reported by genetic analyses that bacteria belonging to the family Enterobacteriaceae, except Klebsiella spp. (1, 2) and Pseudomonas spp., have chromosomally encoded inducible AmpC 3-lactamases (8,14,16,20,26,34) times show resistance to imipenem and broad-spectrum P-lactams (7,17,37). The imipenem resistances of these bacteria are mainly due to the production of metallo 3-lactamases that belong to class B. The class B enzymes require zinc ions for enzyme activity and demonstrate a primary structure quite different from those of the class A and class C enzymes belonging to the group of serine P-lactamases.Recently, several strains of Serratia marcescens were reported to show resistance to carbapenems as well (35). In this study, we clearly show that a clinically isolated strain of S. marcescens (TN9106) showing resistance to imipenem has a chromosomally encoded metallo 3-lactamase gene.

A plasmid-mediated metallo-␤-lactamase gene was cloned from a carbapenem-resistant Serratia marcescens strain, AK9373. The metallo-␤-lactamase gene was identical to the bla IMP , and it was located in the space between an integrase-like gene and an aac(6)-Ib-like gene. The deduced amino acid sequence for the putative integrase gene showed considerable identity (60.9%) to that of the Escherichia coli integrase reported. Sequences similar to the GTTRRRY and an atypical 59-base element containing a 67-bp inverted repeat sequence, which were peculiar to the integrase-dependent recombination, were also conserved in the flanking regions of the bla IMP gene. These findings imply that the metallo-␤-lactamase gene in S. marcescens AK9373 is carried by a novel integron-like element that is mediated by a transferable large plasmid.Carbapenems, such as imipenem, are potent agents for chemotherapy in infectious diseases caused by gram-negative bacteria including the family Enterobacteriaceae, since they are quite stable to hydrolysis by ␤-lactamases produced by these organisms (3, 4, 9, 10, 12). However, several clinical isolates of Bacteroides fragilis, Aeromonas hydrophila, and Pseudomonas aeruginosa were reported to be resistant to these agents because of production of metallo-␤-lactamases (7,13,15,19) belonging to Ambler's class B (1). Recently, we also isolated an imipenem-resistant Serratia marcescens strain, TN9106, and characterized a novel enterobacterial metallo-␤-lactamase, IMP-1, produced by this strain (14). Transfer of carbapenem resistance was observed in some strains of B. fragilis (5) and P. aeruginosa (19), though genetic characterization has not been done yet. In the present study, we investigated the structural features of an element carrying the metallo-␤-lactamase gene mediated by a transferable large plasmid harbored by an imipenem-resistant S. marcescens strain, AK9373 (11).(This study was presented in part at the 34th Interscience Conference on Antimicrobial Agents and Chemotherapy, Orlando, Fla., 4 to 7 October 1994 [2].) Cloning of the imipenem resistance gene. S. marcescensAK9373 showing high resistance to various broad-spectrum ␤-lactams including imipenem was isolated from a patient with a urinary tract infection at a general hospital in Anjyo, Japan, in 1993 (11). The total DNA of this strain was prepared and digested with BamHI; then, the resultant fragments were ligated in plasmid vector pMK16 (14). Escherichia coli HB101 was transformed with these recombinant plasmids, and several colonies grown on Luria-Bertani agar plates supplemented with 8 g of ceftazidime per ml were isolated (14). A 9-kb insert was generally found among the recombinant plasmids harbored by these ceftazidime-resistant transformants. The restriction sites of several endonucleases in the recombinant plasmid pSMB731 were determined as shown in Fig. 1, and the imipenem resistance gene was localized near the SmaI site by deletion analysis. Sequence analyses and identification of ORFs. The BamHISacI fragment was subcloned int...

The distribution of strains producing metallo-␤-lactamase among 105 strains of Serratia marcescens was investigated. All of these strains were isolated in seven general hospitals located in Aichi Prefecture, Japan, from April to May 1993. Southern hybridization analysis suggested that four S. marcescens strains, AK9373, AK9374, AK9385, and AK9391, had a metallo-␤-lactamase genes similar to the bla IMP gene found by our laboratory (E. Osano, Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato, Antimicrob. Agents Chemother. 38:71-78, 1994), and these four strains showed resistance to carbapenems as well as to the other broad-spectrum ␤-lactams. In particular, strains AK9373, AK9374, and AK9391 showed an extraordinarily high-level resistance to imipenem (MICs, м64 g/ml), whereas strain AK9385 demonstrated moderate imipenem resistance (MIC, 8 g/ml). The imipenem resistance of AK9373 was transferred to Escherichia coli CSH2 by conjugation with a frequency of 10 ؊5. The DNA probe of the bla IMP gene hybridized to a large plasmid (approximately 120 kb) transferred into the E. coli transconjugant as well as to the large plasmids harbored by AK9373. On the other hand, although we failed in the conjugational transfer of imipenem resistance from strains AK9374, AK9385, and AK9391 to E. coli CSH2, imipenem resistance was transferred from these strains to E. coli HB101 by transformation. A plasmid (approximately 25 kb) was observed in each transformant which acquired imipenem resistance. The amino acid sequence at the N terminus of the enzyme purified from strain AK9373 was identical to that of the metallo-␤-lactamase IMP-1. In contrast, strains ES9348, AK9386, and AK93101, which were moderately resistant to imipenem (MICs, м4 to Ϲ8 g/ml), had no detectable bla IMP gene. As a conclusion, 19% of clinically isolated S. marcescens strains in Aichi Prefecture, Japan, in 1993 were resistant to imipenem (MICs, Ն2 g/ml), and strains which showed high-level imipenem resistance because of acquisition of a plasmid-mediated bla IMP -like metallo-␤-lactamase gene had already proliferated as nosocomial infections, at least in a general hospital.

The genomic organization of the chromosomal cps region that is responsible for capsular polysaccharide synthesis in Klebsiella pneumoniae Chedid (O1:K2) was investigated. Deletion analyses and Southern hybridization studies suggested that the central region of the cloned 29-kb BamHI fragment is indispensable for K2 capsular polysaccharide synthesis. The 24,329-bp nucleotide sequence of the Klebsiella cps region was determined and deposited in the EMBL and GenBank databases through DDBJ and assigned accession number D21242. Nineteen possible open reading frames (ORFs) were identified in the sequenced area. Among them, 13 ORFs are very close to each other. Six of the 19 ORFs show considerable nucleotide sequence similarities to Salmonella typhimurium cpsG, cpsB, rfbP, and orf2.8, Escherichia coli gnd, and Haemophilus influenzae bexD, respectively. Moreover, the deduced amino acid sequence of the ORF10 product demonstrated a highly hydrophobic profile and showed putative membrane topology similarity to Rickettsia prowazekii ATP/ADP translocase. Nucleotide sequences similar to the 54 -dependent promoter, as well as the usual ؊35 and ؊10 sequences, were identified just upstream of ORF3, which is the first ORF in the polycistronic structure. Furthermore, a sequence (GGGCGGTAGCGT) found just downstream of the 54 -dependent promoter-like sequence was generally conserved among gene clusters implicated in cell surface polysaccharide synthesis, such as Salmonella rfb and viaB and E. coli kpsMT and rfaQPG. A possible transcriptional terminator with a hairpin loop structure found just downstream of ORF15 that is a homolog of E. coli gnd. K2 capsular polysaccharide biosynthesis in E. coli K-12 depends on cpsB (mannose-1-phosphate guanyltransferase gene), and Klebsiella cpsB, found in the downstream region of the polycistronic structure, was able to complement cpsB of E. coli. Results of transposon insertion and promoter-cloning analyses were consistent with the results of nucleotide sequence analysis.

We applied PCR to the rapid detection of the metallo-␤-lactamase gene, bla IMP , in clinically isolated gram-negative rods. A total of 54 high-level ceftazidime-resistant strains (MICs, >128 g/ml) were subjected to PCR analyses with the bla IMP-specific primers, since the bla IMP-bearing clinical isolates tested in our previous study always demonstrated high-level resistance to ceftazidime. Twenty-two bla IMP-positive strains including 9 Pseudomonas aeruginosa, 9 Serratia marcescens, 2 Alcaligenes xylosoxidans, 1 Pseudomonas putida, and 1 Klebsiella pneumoniae strains were newly identified from 18 different hospitals in Japan. These strains were mostly isolated from urine samples and showed high-level resistance to almost every cephem, while their levels of resistance to carbapenems were diverse. The PCR analyses with novel integrase gene-specific (intI3) and acc(6)-Ib gene-specific primers suggested that the integron structure found in a large plasmid harbored by S. marcescens AK9373 was also well conserved among bla IMP-positive strains. These results imply that the bla IMP gene cassettes have been dispersing into various gram-negative rods with the help of the newly identified integron element. Thus, the PCR-aided rapid detection will be helpful for the early recognition of emerging bla IMP-positive clinical isolates which demonstrate consistent resistance to ␤-lactams.