Koji O'hara

Amino Acid Substitutions in a Variant of IMP-1 Metallo-β-Lactamase

ABSTRACT In the course of surveying for the carbapenem-hydrolyzing metallo-β-lactamase gene blaIMP in pathogenic bacteria by the PCR method, we detected a gene encoding a variant metallo-β-lactamase, designated IMP-3, which differed from IMP-1 by having low hydrolyzing activity for penicillins and carbapenems. PCR product direct sequencing of a 2.2-kb segment revealed that the geneblaIMP-3 was located on a cassette inserted within a class I integron in the pMS390 plasmid. The 741-bp nucleotide sequence of blaIMP-3 was identical to that ofblaIMP-1, except for seven base substitutions. Among these were two, at nucleotide positions 314 and 640, which caused amino acid alterations. Hybrid bla genes were constructed from blaIMP-3 andblaIMP-1 by recombinant DNA techniques, and β-lactamases encoded by these genes were compared with those of the parents IMP-3 and IMP-1 under the same experimental conditions. The kinetic parameters indicated that the inefficient hydrolysis of benzylpenicillin, ampicillin, imipenem, and ceftazidime by IMP-3 was due to the substitution of glycine for serine at amino acid residue 196 in the mature enzyme. This alteration corresponded to the presence of guanine instead of an adenine at nucleotide position 640 of theblaIMP-3 gene. This indicated that extension of the substrate profile in the metallo-β-lactamase IMP-1 compared to IMP-3 is the result of a one-step single-base mutation, suggesting that the gene blaIMP-3 is an ancestor ofblaIMP-1.

Detection of a Variant Metallo-β-Lactamase, IMP-10, from Two Unrelated Strains of Pseudomonas aeruginosa and an Alcaligenes xylosoxidans Strain

ABSTRACT The gene blaIMP-10 of a variant metallo-β-lactamase, IMP-10, had a single base replacement of G by T at nucleotide 145, which led to an amino acid alteration of Val49 to Phe compared to the IMP-1 enzyme, indicating that IMP-10 was a point mutation derivative of IMP-1. Highly purified enzymes revealed that IMP-10 was different from IMP-1 in its extremely low hydrolyzing activities for penicillins, such as benzylpenicillin, ampicillin, and piperacillin.

Functional Analysis of the Active Site of a Metallo-β-Lactamase Proliferating in Japan

ABSTRACT An R-plasmid-mediated metallo-β-lactamase was found inKlebsiella pneumoniae DK4 isolated in Japan in 1991. The nucleotide sequence of its structural gene revealed that the β-lactamase termed DK4 was identical to the IMP-1 metallo-β-lactamase which was mediated by a chromosomal gene ofSerratia marcescens TN9106 isolated in Japan in 1991 (E. Osano et al., Antimicrob. Agents Chemother. 38:71–78, 1994). The dose effect of DK4 β-lactamase production on the resistance levels indicated a significant contribution of the enzyme to bacterial resistance to all the β-lactams except monobactams. The enzymatic characteristics of the DK4 β-lactamase and its kinetic parameters for nine β-lactams were examined. The DK4 β-lactamase was confirmed to contain 2 mol of zinc per mol of enzyme protein. The apoenzyme that lacked the two zincs was structurally unstable, and the activities of only 30% of the apoenzyme molecules could be restored by the addition of 1 mM zinc sulfate. The substitution of five conserved histidines (His28, His86, His88, His149, His210) and a cysteine (Cys168) for an alanine indicated that His86, His88, and His149 served as ligands to one of the zincs and that Cys168 played a role as a ligand to the second zinc. Both zinc molecules contribute to the enzymatic process. Mutant enzymes that lack only one of these retained some activity. Additionally, a conserved aspartic acid at position 90 was replaced by asparagine. This mutant enzyme showed an approximately 1,000 times lower kcat value for cephalothin than that of the wild-type enzyme but retained the two zincs even after dialysis against zinc-free buffer. The observed effect of pH on the activity suggested that Asp90 functions as a general base in the enzymatic process.

Enzymatic Inactivation of a New Aminoglycoside Antibiotic, Sisomicin, by Resistant Strains of Pseudomonas aeruginosa

The antibacterial activity of sisomicin (SS), a new aminoglycoside antibiotic active toward clinical isolates of Pseudomonas aeruginosa, was determined and compared with that of the gentamicin C complex. Both drugs were effective against these strains and showed almost the same antibacterial activity. A few strains were found to be resistant to SS. The antibiotic was inactivated by a cell-free extract from the SS-resistant strains due to acetylation of the drug. Comparative studies of the inactivation of the drugs which lack a 6′-amino group in the amino sugar linked to 2-deoxystreptamine strongly suggested that SS inactivation was due to acetylation of the 6′-amino group of the 4′,5′-didehydropurpurosamine moiety.

Novel Fosfomycin Resistance of Pseudomonas aeruginosa Clinical Isolates Recovered in Japan in 1996

The antibiotic fosfomycin has been used in Japan for 21 years, and although it can be used as a single agent, it is more active when used in combination with various other antibiotics (3). Fosfomycin enters the cells of fosfomycin-susceptible bacteria by means of two different transport uptake systems: GlpT and UhpT (10). In our studies, we measured the MICs of fosfomycin for 412 randomly selected Pseudomonas aeruginosa clinical isolates, collected in 1996 from across Japan, and investigated a new mechanism of resistance found among fosfomycin-resistant isolates. Fosfomycin MICs were determined by agar dilution with an inoculum of 500 cells to the nutrient agar surface (Difco Laboratories) (4, 9). The enzymatic inactivation of fosfomycin using crude extracts with and without cofactor (40 mM ATP) was determined by measuring residual fosfomycin (9). The transfer frequency of fosfomycin resistance was determined as described previously (9), using P. aeruginosa PAO2142Rp (8) as recipient. The distribution of MICs of fosfomycin for the 412 isolates of P. aeruginosa exhibited a cluster of the majority of strains centered around an MIC value of 6.25 μg/ml and a second significant cluster centered at MICs of more than 12,800 μg/ml. Compared with the 1975 report of Goto et al. (4), our results suggested that the susceptibility of P. aeruginosa to fosfomycin has remained almost unchanged for 21 years. In 1977, although 65% of 60 fosfomycin-resistant gram-negative isolates transferred some other demonstrable antibiotic resistance to Escherichia coli K-12 in 1977 (2), there were no occurrences of the transfer of fosfomycin resistance. Of the 67 fosfomycin-resistant isolates in our study with MICs greater than 800 μg/ml, none transferred this resistance to the recipient strain PAO2142Rp. Thus, our data suggest that transferable plasmid-encoded fosfomycin resistance has not emerged even in recent Japanese P. aeruginosa isolates. Crude extracts from two fosfomycin-resistant isolates, CU252 and CU358, completely inactivated fosfomycin but only in the presence of ATP (Fig. ​(Fig.11). FIG. 1 Inactivation of fosfomycin by crude extracts of P. aeruginosa CU252 and CU358 with and without ATP. ●, heated crude extract (CU252); ■, crude extract (CU252) in the presence of ATP; ▴, crude extract (CU252) without ATP; ○, ... Fosfomycin resistance in clinical isolates is caused mainly by an alteration of the chromosomally encoded GlpT transport system. The plasmid-mediated fosfomycin glutathione S-transferase genes fosA and fosB, found in only a low percentage of strains (1, 6, 7), catalyze the addition of glutathione to fosfomycin (10). The fosfomycin inactivation mechanism reported here appears to be new, as it was nontransferable and ATP dependent. It will be interesting to compare it with the mechanism in fosC and in fomA and fomB cloned into E. coli from fosfomycin-producing Pseudomonas syringae and Streptomyces wedmorensis (5), respectively. The correlation between fosfomycin resistance of P. aeruginosa and the mechanism of resistance, including enzyme characterization, will be the subject of our future studies.

Mechanism of resistance to some cephalosporins in Staphylococcus aureus.

The mechanism of resistance to some cephalosporins in Staphylococcus aureus strains was investigated with high-pressure liquid chromatography and nuclear magnetic resonance spectrometry. Drug inactivation by penicillinase was found to be the main mechanism of resistance to cefazolin, cephaloridine, and cephalothin in S. aureus.

Microbial degradation of cephalothin by cephalothin-susceptible Escherichia coli.

Cephalothin (CET)-susceptible Escherichia coli, which can degrade CET after prolonged incubation in broth containing a concentration of the drug greater than the minimum inhibitory concentration, was found in a clinical specimen. The substrate specificity of the partially purified enzyme to cephalosporin analogs strongly indicated the occurrence of CET-specific degradation. Nuclear magnetic resonance analysis of the degradation reaction demonstrated the appearance of two new signals attributed to deacetyl CET. This suggests the possibility of the presence of acylesterase.

Prevalence of R factors in Pseudonomas aeruginosa.

There are many reports of R factors in Pseudomonas aeruginosa (Roe, Jones & Lowbury, 1971 ; Witchitz & Chabbert, 1971 ; Bryan, Van den Elzen & Tseng, 1972; Grinsted et al. 1972; Olsen & Shipley, 1973; Iyobe et al. 1974; Van Rensburg & de Kock, 1974) but there has been little work on the prevalence of R factors in clinical isolates of P. aeruginosa (see Holloway & Richmond, 1973). We have surveyed the drug resistances of 89 distinct strains of P. aeruginosa isolated from patients suffering from urinary tract infections and have found that 10 (I 1.2 %) of the isolates transfer one or more of their resistance determinants to drug-sensitive recipients.

Relationship Between Chloramphenicol Acetyltransferase Activity and the Number of Resistance Genes

Various mutants of an Escherichia coli K-12 strain were prepared, in which a chloramphenicol (CM) resistance gene (cml) derived from an R factor, R100-1, was integrated into the chromosome. The CM acetyltransferase (CATase) activity of these strains and the strain carrying R100-1 were determined during exponential growth with the following results. (i) The CATase activity varied, depending upon the site of integration of the cml gene on the chromosome. Activity was found to be higher when the cml gene was integrated nearer the replication origin of the chromosome, the total enzyme activity found was the sum of activities coded by each gene separately. (iii) When the cml gene was in a cytoplasmic state on an R factor, R100-1, the expressed enzyme activity was four-to eightfold higher than that in the chromosomal state, suggesting the existence of about four to eight copies of R factor per chromosome. The CATase activity returned to the level of that expressed by R100-1 when the chromosomal cml gene was detached and picked up by an R factor.

Nuclear magnetic resonance spectrometric assay of beta-lactamase.

Beta-Lactam antibiotics and the crude enzyme were mixed in deuterium oxide and placed in a nuclear magnetic resonance tube. The change of the nuclear magnetic resonance spectrum during the enzymatic reaction was then analyzed to determine beta-lactamase activity. By using beta-lactam antibiotics such as penicillins, cephalosporins, and cephamycins as substrates, a comparison of the beta-lactamase activities was made between the nuclear magnetic resonance spectrometric assay and the iodometric assay. There was a close correlation between these two methods.