Sensitivity of 5 beta-lactam antibiotics to beta-lactamase isolated from E. coli and its respective transconjugates

54 beta-lactamase producing E. coli were tested to observe their eventual capacity to transfer beta-lactamase production by conjugation to a receiving E. coli K12 C600 Na-. About 16% (9/54) of these strains transferred beta-lactamase producing capacity. MICs of five beta-lactam antibiotics (Ampicillin, Cephaloridine, Cephalexine, Cefuroxime, Cefotaxime) were performed against E. coli donors and E. coli K12 C600 transconjugates. It was observed a remarkable increase only of Ampicillin MICs against all transconjugates++. Beta-lactamases produced by donors and transconjugants were isolated and purified by sonication and high speed centrifugation. Sensitivity of the six antibiotics to these purified beta-lactamases was assessed by a spectrophotometric method that utilizes the velocity of cytochrome c reduction. beta-lactamases produced by transconjugants have identical substrate profile that beta-lactamases produced by donors.     

Purification and characterization of a beta-lactamase from Haemophilus ducreyi in Escherichia coli

A pCb plasmid encoding a beta-lactamase from Haemophilus ducreyi was transferred to Escherichia coli, purified, and characterized. The beta-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and chelating Sepharose fast flow loaded with Zn(2+). The purified enzyme resulted in a major band at approximately 30-kDa on SDS-PAGE and its pI was determined to be 5.4. The beta-lactamase could hydrolyze both penicillin antibiotics including ampicillin, benzylpenicillin, and carbenicillin as well as cephalosporin antibiotics including nitrocefin, cephalothin, cephaloridine, and cefoperazone. However, benzylpenicillin was the best substrate. The enzyme activity was inhibited by clavulanic acid but not by boric acid, cefotaxime, ethylenediaminetetraacetic acid, or phenylmethylsulfonyl fluoride. The sequence of the beta-lactamase gene was also determined. It confirmed that the enzyme belonged to a class A beta-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322. Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino-acid sequence. The valine at position 82 (ABL 84) was changed to isoleucine and the alanine at position 182 (ABL 184) was changed to valine. Genetic homogeneity among beta-lactamases is remarkable. Amino acid sequencing of some beta-lactamases has shown that substitution of only a few amino acids in the bla gene leads to high-level resistance against specific cephalosporins.     

Beta-lactamases with a wide substrate spectrum in gram negative strictly anaerobic rods

This study was performed to determine the susceptibility of the clinical strains of Gram-negative strictly anaerobic rods to newer beta-lactam antibiotics. Also, the trial was undertaken to detect strains producing extended-spectrum beta-lactamases (ESBLs) and inducible beta-lactamases (IBLs) among Bacteroides spp. and Prevotella spp. rods isolated from hospitalized patients. One hundred strains of Gram-negative, obligatory anaerobic rods were applied in the study. The strains were identified in automatic ATB system using API 20 A strips. beta-lactamase-positive strains were determined with disc nitrocefin test. ESBL-producing strains were detected with double disc test according to Jarlier et al. (1988). Clavulanate was applied as the inhibitor of these beta-lactamases (AMO/CLAV disc). ESBL-positive strains were confirmed with the use of E test (TZ/TZL strip). Inducible beta-lactamases were determined by double disc method according to Sanders and Sanders (1979). Cefoxitin was the inducer of these beta-lactamases (FOX disc). Among 93 Bacteroides spp. strains and 7 Prevotella spp. strains, 91 strains (91%) produced beta-lactamases. Two ESBL-producing strains (2%) were detected. Strains producing inducible beta-lactamases (IBL) were not found. A high activity of the examined beta-lactam antibiotics against strains of Gram-negative anaerobes was found. The majority of strains were susceptible to piperacillin (95%), piperacillin combined with tazobactam (99%), ticarcillin combined with clavulanic acid (99%), meropenem (97%) and imipenem (99%). The obtained results indicate the necessity of ESBL determination among strains of the genus Bacteroides, isolated from clinical specimens. Newer beta-lactam antibiotics, especially penicillins in combination with beta-lactamase inhibitors and carbapenems, are useful in empiric therapy of infections caused by Bacteroides spp. and Prevotella spp. anaerobic rods.     

Simultaneous production of two types of beta-lactamase in Escherichia coli and Providencia stuartii.

The production of beta-lactamase has been studied in two strains isolated from clinical samples: Providencia stuartii MULB 501 and Escherichia coli MULB 130. These strains were selected for their high resistance level to penicillins and cephalosporins. Determination and identification of beta-lactamase activity were achieved by combining several up-to-date methods including (i) neutralization by anti-beta-lactamase sera, (ii) purification by affinity chromatography on cephalosporin C linked Sepharose 4B, (iii) determination of substrate specificity and kinetic values (Km and Vmax) by a computerized microacidimetric method, and (iv) isoelectric focusing. The results clearly demonstrate that in these two strains there is a simultaneous production of different beta-lactamases: the first one is similar to the TEM penicillinase and the second one shares a typical cephalosporinase profile. This double beta-lactamase production is a relatively rare phenomenon.     

Susceptibility and molecular mechanisms of resistance to cephalosporins of Escherichia coli strains isolated from patients with nosocomial infections

Resistance to wide spectrum of antibiotics was studied and most widespread genetic determinants of resistance to beta-lactam antibiotics were revealed. Susceptibility testing was performed using serial broth microdilution method. Detection of class A expanded spectrum beta-lactamases genes (TEM, SHV, CTX) by polymerase-chain reaction method was performed in 90 strains. Carbapenems remained the most active antibacterial agents with respect to studied E. coli strains. Among the 3rd generation cephalosporins the lowest minimal inhibitory concentrations were observed for inhibition-protected combined agents (ceftazidime/clavulanic acid and cefoperazone/ sulbactam). Alone or in various combinations TEM, SHV, and CTX types of beta-lactamases were found in 58.9%, 14.4%, and 77.8% of strains. Combinations of 2 determinants were detected in 55.6% of the isolates, and all 3 determinants--in 5.6%. Most often E. coli was isolated in patients with urinary tract infections. Carbapenems and inhibition-protected combined 3rd generation cephalosporins are the most active agents against E. coli.     

Purification and properties of beta-lactamase from Proteus morganii.

The cephalosporin beta-lactamase was purified from a strain of Proteus morganii that showed resistance to beta-lactam antibiotics and produced the enzyme constitutively. The purified enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis and consisted of a single polypeptide of molecular weight 38,000 to 40,000 from gel filtration of Sephadex G-100 and sodium dodecyl sulfate-acrylamide gel electrophoresis, its isoelectric point being pH 7.2 No cysteine residue was found in its amino acid composition. The specific activity was 190 mumol/min per mg of the purified enzyme protein for the hydrolysis of cephaloridine, the optimal pH was about 8.5 and the optimal temperature was 50 degrees C. Antibodies against the purified beta-lactamase inhibited not only the enzyme activity of the purified preparation, but also the enzyme activity of all of the other strains of P. morganii so far tested, regardless of whether the modes of their production were inducible or constitutive. None of the beta-lactamases produced by beta-lactam antibiotic-resistant strains of other species of Proteus was affected at all by the antibodies, thus showing that the purified cephalosporin beta-lactamase was of the species-specific type. The enzymological properties of the preparation have been compared with those of beta-lactamases derived from other gram-negative enteric bacteria.     

Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii,Citrobacter murliniae,Citrobacter werkmanii,Escherichia fergusonii and Enterobacter cancerogenus

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.     

Characterization of a new beta-lactamase gene from isolates of Vibrio spp. in Korea

PCR was performed to analyze the beta-lactamase genes carried by ampicillin-resistant Vibrio spp. strains isolated from marine environments in Korea between 2006 and 2009. All 36 strains tested showed negative results in PCR with the primers designed from the nucleotide sequences of various known beta-lactamase genes. This prompted us to screen new beta-lactamase genes. A novel beta-lactamase gene was cloned from Vibrio alginolyticus KV3 isolated from the aquaculture water of Geoje Island of Korea. The determined nucleotide sequence (VAK-3 beta-lactamase) revealed an open reading frame (ORF) of 852 bp, encoding a protein of 283 amino acids (aa), which displayed low homology to any other beta-lactamase genes reported in public databases. The deduced 283 aa sequence of VAK-3, consisting of a 19 aa signal peptide and a 264 aa mature protein, contained highly conserved peptide segments specific to class A beta-lactamases including the specific amino acid residues STFK (62-65), SDN (122-124), E (158), and RTG (226-228). Results from PCR performed with primers specific to the VAK-3 beta-lactamase gene identified 3 of the 36 isolated strains as V. alginolyticus, Vibrio cholerae, and Photobacterium damselae subsp. damselae, indicating the utilization of various beta-lactamase genes including unidentified ones in ampicillin-resistant Vibrio spp. strains from the marine environment. In a mating experiment, none of the isolates transfered the VAK-3 beta-lactamase gene to the Escherichia coli recipient. This lack of mobility, and the presence of a chromosomal acyl-CoA flanking sequence upstream of the VAK-3 beta- lactamase gene, led to the assumption that the location of this new beta-lactamase gene was in the chromosome, rather than the mobile plasmid. Antibiotic susceptibility of VAK-3 beta-lactamase was indicated by elevated levels of resistance to penicillins, but not to cephalosporins in the wild type and E. coli harboring recombinant plasmid pKV-3, compared with those of the host strain alone. Phylogenetic analysis showed that VAK-3 beta-lactamase is a new and separate member of class A beta-lactamases.     

Sensitivity to selected beta-lactam antibiotics of clinical strains of Escherichia coli and Klebsiella pneumoniae

The studies aimed at analysing the resistance to some beta-lactam antibiotics among E. coli and K. pneumoniae clinical isolates and at evaluating. The extended spectrum of beta-lactamases (ESBL) production in the isolates. The analysis included 137 E. coli strains and 52 K. pneumoniae strains, isolated from hospitalized patients and out-patients treated in the first trimester of 1998. The strains were identified using the ATB computer system. Antibiotic sensitivity of the isolates was determined by disc-diffusion tests. ESBL production capacity of E. coli and K. pneumoniae strains was estimated by double-disc and ATB BLSA tests. Most of the analysed E. coli strains were found to exhibit significant sensitivity to compound penicillin preparations containing beta-lactam inhibitor (Augmentin, Tazocin) and to the third generation cefalosporins, in contrast, K. pneumoniae strains much more frequently were resistant to the drugs. Among the obtained isolates, 3 (2.2%) E. coli strains and 21 (40.4%) K. pneumoniae strains produced ESBL but all the isolates proved sensitive to imipenem. In evaluation of ESBL production-detecting tests, the double-disc test was found to be more reliable than ATB BLSA test.     

Inhibition of the β-lactamases of Escherichia coli and Klebsiella aerogenes by semi-synthetic penicillins

1. A new automated micro-iodometric method is described for screening compounds for inhibitory action against beta-lactamase enzymes. 2. Over 1000 semi-synthetic penicillins were tested for inhibitory activity against the beta-lactamase of Escherichia coli B11 and 18 showed a fractional inhibition similar to or higher than that of methicillin. 3. The best inhibitors were alkoxy- and halogen-substituted phenyl-, naphthyl- or quinolyl-penicillins. 2-Isopropoxy-1-naphthylpenicillin (BRL 1437) was clearly the best and had a K(i) value about 1% of that of methicillin. 4. The inhibition of the beta-lactamase of E. coli B11 by BRL 1437 was shown to be reversible and competitive. The K(i) was 0.004mum and K(i)/K(m) with ampicillin and p-hydroxyampicillin (BRL 2333) was about 0.0001. The K(m) and V(max.) values were determined for the beta-lactamases of E. coli B11 and Klebsiella aerogenes A against a variety of penicillins. Cell-bound and solubilized enzymes gave similar K(i) and K(m) values. 5. BRL 1437 was superior to cloxacillin and methicillin for inhibition of the beta-lactamase of live, fully grown cultures of several strains of E. coli and K. aerogenes. Of a group of inhibitors BRL 1437 was the most stable to the beta-lactamase of E. coli B11.     

In vitro activity of three different antimicrobial agents against ESBL producing Escherichia coli and Klebsiella pneumoniae blood isolates

Extended spectrum beta-lactamases (ESBLs) usually associated with multiple drug resistance, including beta-lactam and non-beta-lactam antibiotics. This resistance can cause Limitation in the choice of drugs appropriate for using in clinical practice, especially in life-threatening infections. In this study we aimed to investigate in vitro activity of meropenem, ciprofloxacine and amikacin against ESBL-producing and non-producing blood isolates of Escherichia coli and Klebsiella pneumoniae strains. Fifty-eight E. coli (21 ESBL-producing, 37 non-ESBL producing) and 99 K. pneumoniae (54 ESBL-producing, 45 non-ESBL producing) strains were included in the study. The presence of ESBL was investigated by double disk synergy test and E-test methods. Antibiotic susceptibility test was done by microdilution method according to NCCLS guideline. In vitro susceptibilities of ESBL producing E. coli and K. pneumoniae strains were found as 100% for meropenem, 33.3% and 25.9% for ciprofloxacine, 94.5% and 83.3% for amikacin. It was observed that; meropenem was equally active agent in both ESBL-producing and non-producing strains, and its activity was not affected by ESBL production. Whereas amikacin activity was minimally affected and ciprofloxacine activity was markedly decreased by ESBL production. In conclusion, meropenem seems to be better choice of antibiotic should be used for ESBL positive life-threatening infections, because of remaining highest activity.     

Mutagenic effect of acridine orange on the expression of penicillin G acylase and beta-lactamase in Escherichia coli

AIMS: The present work aimed to improve the production of penicillin G acylase (PGA) and reduce the beta-lactamase activity through acridine orange (AO) induced mutation in Escherichia coli. METHODS AND RESULTS: Three wild E. coli strains BDCS-N-FMu10, BDCS-N-S21 and BDCS-N-W50, producing both the enzymes PGA and beta-lactamase were treated by AO. Minimum inhibitory concentration of AO was 10 microg ml(-1) and it was noted that bacterial growth was gradually suppressed by increasing the concentration of AO from 10 to 100 microg ml(-1). The highest concentration that gave permissible growth rate was 50 microg ml(-1). The isolated survivals were screened on the bases of PGA and beta-lactamase activities. Among the retained mutants, the occurrence of beta-lactamase deficient ones (91%) was significantly higher than penicillin acylase deficient ones (27%). CONCLUSIONS: In seven of the mutants, PGA activity was enhanced with considerable decrease in beta-lactamase activity. One of the mutant strains (BDCS-N-M36) exhibited very negligible expression of beta-lactamase activity and twofold increase in PGA activity [12.7 mg 6-amino-penicillanic acid (6-APA) h(-1) mg(-1) wet cells] compared with that in the wild-type strain (6.3 mg 6-APA h(-1) mg(-1) wet cells). SIGNIFICANCE AND IMPACT OF THE STUDY: The treatment of E. coli cells with AO resulted in mutants with enhanced production of PGA and inactivation of beta-lactamase. These mutants could be used for industrial production of PGA.     

Exploring the effectiveness of tazobactam against ceftazidime resistant Escherichia coli: insights from the comparison between susceptibility testing and beta-lactamase inhibition

Thirteen clinical isolates of Escherichia coli resistant to ceftazidime that possessed an AmpC and other (beta-lactamases were identified. The effectiveness of different formulations of piperacillin/tazobactam to other beta-lactams was compared. Antibiotic susceptibility testing, polymerase chain reaction, amplification of blaTEM, blaSHV and blaAmpC, and enzyme-linked immunosorbent assays to identify AmpC beta-lactamases were performed. Hydrolysis rates were obtained and residual enzymatic activity was determined. Cefepime and ertapenem were more active than piperacillin/tazobactam. In contrast, increasing the relative proportion of tazobactam improved susceptibility testing. Twenty micromolar tazobactam inhibited total beta-lactamase activity (as measured by nitrocefin hydrolysis rates) by greater than 75% against all isolates tested: in 11 of 13 E. coli isolates, total beta-lactamase activity was inhibited by 90%. The observed differences between MIC determinations and susceptibility to enzymatic inactivation by tazobactam against E. coli containing AmpC and other -lactamases may be due to the final tazobactam concentration achieved in the periplasmic space. Factors determining this are critical considerations in assessing beta-lactamase inhibitor potency.     

Antimicrobial susceptibility and molecular mechanisms of resistance to beta-lactams of gram-negative microorganisms--causative agents of nosocomial infections

Profiles and mechanisms of resistance to beta-lactam antibiotics of isolates of Gram-negative microorganisms, which are causative agents of infections in Intensive Care Unit of hospital surgery department, were studied. Two hundred and ten clinical isolates were studied: Pseudomonas aeruginosa--86 strains (40.9%), Acinetobacter baummanii--45 strains (21.4%), Klebsiella pneumoniae--52 strains (24.8%), Escherichia coli--23 strains (11%), Enterobacter spp.--4 strains (1.9%). Profiles of antibiotic resistance were studied by the method of serial microdilutions; detection of most widespread and clinically significant genes of beta-lactamases of Gram-negative bacteria was performed by polymerase chain reaction. Carbapenems and cefoperazone/sulbactam were the most active antibiotics. Local features of distribution of beta-lactamase coding genes (TEM, SHV, CTX) in K. pneumoniae and E. coli isolates were revealed. Eleven strains of P. aeruginosa resistant to carbapenems and possessing genetic determinants of VIM-group, which codes metallo-beta-lactamases, were isolated. Obtained data allows to assess the parameters of resistance to beta-lactam antibiotics and to reveal the main mechanisms of such resistance in etiologic agents of nosocomial infections, that, in its turn, allows to choose preparations for etiotropic therapy.     

Prevalence of extended-spectrum beta-lactamases produced by nosocomial isolates of Enterobacteriaceae in Trakya University Hospital,Turkey

The prevalence of extended-spectrum beta-lactamase (ESBL) production by 194 nosocomial isolates of Enterobacteriacea recovered from 1995 to 1999 was investigated. The ESBL production was determined by the double-disk synergy test and was confirmed by the E-test ESBL strip. Twenty-three isolates (21 Klebsiella pneumoniae, one Escherichia coli, one Providencia rettgeri) were found as ESBL-producers (11.8%). These isolates were also usually resistant to non-betalactam antibiotics. Most of them contained a beta-lactamase with a pI of 7.6. All the strains conjugally transferred their ESBLs to recipient E. coli. Contrary to others, ESBL-producing K. pneumoniae strains isolated in 1999 were resistant to ciprofloxacin, and had the identical plasmid profiles suggestive of an outbreak. Ciprofloxacin resistance in these strains could not be transferred. In conclusion, K. pneumoniae was the main ESBL-producing species among nosocomial isolates of Enterobacteriacae in our hospital.     

Effect of β-Lactamase Location in Escherichia coli on Penicillin Synergy

Resistance to ampicillin in Escherichia coli is due generally to the presence of a beta-lactamase (penicillinase). Resistant strains have been found to fall into two groups: those with high-level resistance (1,000 mug/ml or greater) and those with low-level resistance (8 to 250 mug/ml). Most of the high-level resistant organisms posses beta-lactamases whose synthesis is episomally mediated. These strains release penicillinase from the cell when they are subjected to osmotic shock. Low-level resistant strains do not release the enzyme with osmotic shock. High-level resistant strains are not susceptible to the synergistic action of a penicillinase-resistant penicillin with ampicillin. Seventy eight per cent of low-level resistant strains are susceptible to the synergistic action of ampicillin and oxacillin. The two types of beta-lactamases are similar in regard to most properties; both enzymes are subject to competitive inhibition by penicillinase-resistant penicillins. The difference in location in the cell might explain why only some strains of E. coli are susceptible to the synergistic action of penicillin combinations.     

Sanfetrinem, the member of trinems--in vitro activity against Klebsiella pneumoniae producing ESBL

Sanfetrinem is the first member of tricyclic beta-lactams (trinems) which can be administered orally as a hexatil ester. His chemical structure is related to carbapenems. High stability to many beta-lactamases and human renal dehydropeptydase were described. The investigation was performed on 43 strains of Klebsiella pneumoniae producing ESBL isolated from hospitalized patients. The MICs of sanfetrinem and imipenem were determined by E-test. Additionaly, susceptibility to antibiotics was tested by disc diffusion method. Klebsiella pneumoniae ATCC 700603, Escherichia coli ATCC 25922 and Escherichia coli ATCC 35218 were used as a control strains. MIC50 and MIC90 of sanfetrinem and imipenem amounted respectively 0,38/3 mg/1 and 0,19/0,25 mg/l. Range of MIC value was from 0,064 mg/l to 4 mg/l for sanfetrinem, and from 0,094 mg/l to 0,38 mg/l for imipenem. Additionaly, geometric and aritmetic means were calculated to both antibiotics. All results of study were compared using correlation factor and "Student" t-test. None of these 43 Klebsiella pneumoniae strains was resistant to imipenem and cefepime. Majority of isolates demonstrated susceptibility to ciprofloxacin and cefoxitin--90,7% and 74,4% respectively All strains were resistant to gentamicin, amikacin and trimethoprim/sulfamethoxazole.     

Effect of disulfide-bond introduction on the activity and stability of the extended-spectrum class A beta-lactamase Toho-1

The production of class A beta-lactamases is a major cause of clinical resistance to beta-lactam antibiotics. Some of class A beta-lactamases are known to have a disulfide bridge. Both narrow spectrum and extended spectrum beta-lactamases of TEM and the SHV enzymes possess a disulfide bond between Cys77 and Cys123, and the enzymes with carbapenem-hydrolyzing activity have a well-conserved disulfide bridge between Cys69 and Cys238. We produced A77C/G123C mutant of the extended-spectrum beta-lactamase Toho-1 in order to introduce a disulfide bond between the cysteine residues at positions 77 and 123. The result of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) titrations confirmed formation of a new disulfide bridge in the mutant. The results of irreversible heat inactivation and circular dichroism (CD) melting experiments indicated that the disulfide bridge stabilized the enzyme significantly. Though kinetic analysis indicated that the catalytic properties of the mutant were quite similar to those of the wild-type enzyme, E. coli producing this mutant showed drug resistance significantly higher than E. coli producing the wild-type enzyme. We speculate that the stability of the enzymes provided by the disulfide bond may explain the wide distribution of TEM and SHV derivatives and explain how various mutations can cause broadened substrate specificity without loss of stability.     

Isolation and characterization of a beta-lactamase-inhibitory protein from Streptomyces clavuligerus and cloning and analysis of the corresponding gene.

Culture filtrates of Streptomyces clavuligerus contain a proteinaceous beta-lactamase inhibitor (BLIP) in addition to a variety of beta-lactam compounds. BLIP was first detected by its ability to inhibit Bactopenase, a penicillinase derived from Bacillus cereus, but it has also been shown to inhibit the plasmid pUC- and chromosomally mediated beta-lactamases of Escherichia coli. BLIP showed no inhibitory effect against Enterobacter cloacae beta-lactamase, and it also showed no activity against an alternative source of B. cereus penicillinase. BLIP was purified to homogeneity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a size estimate for BLIP of 16,900 to 18,000. The interaction between purified BLIP and the E. coli(pUC) beta-lactamase was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determined to be noncovalent, with an estimated 1:1 molar stoichiometry. The BLIP gene was isolated on a 13.5-kilobase fragment of S. clavuligerus chromosomal DNA which did not overlap a 40-kilobase region of DNA known to contain genes for beta-lactam antibiotic biosynthesis. The gene encoded a mature protein with a deduced amino acid sequence of 165 residues (calculated molecular weight of 17,523) and also encoded a 36-amino-acid signal sequence. No significant sequence similarity to BLIP was found by pairwise comparisons using various protein and nucleotide sequence data banks or by hybridization experiments, and no BLIP activity was detected in the culture supernatants of other Streptomyces spp.