Pseudomonas aeruginosa isolates from patients with cystic fibrosis have different beta-lactamase expression phenotypes but are homogeneous in the ampC-ampR genetic region.

Pseudomonas aeruginosa isolates from 1 of 17 cystic fibrosis patients produced secondary beta-lactamase in addition to the ampC beta-lactamase. Isolates were grouped into three beta-lactamase expression phenotypes: (i) beta-lactam sensitive, low basal levels and inducible beta-lactamase production; (ii) beta-lactam resistant, moderate basal levels and hyperinducible beta-lactamase production; (iii) beta-lactam resistant, high basal levels and constitutive beta-lactamase production. Apart from a base substitution in the ampR-ampC intergenic region of an isolate with moderate-basal-level and hyperinducible beta-lactamase production, sensitive and resistant strains were identical in their ampC-ampR genetic regions. Thus, enhanced beta-lactamase expression is due to mutations in regulatory proteins other than AmpR.     

Molecular and biochemical analysis of AST-1, a class A beta-lactamase from Nocardia asteroides sensu stricto

A beta-lactamase gene was cloned from a Nocardia asteroides sensu stricto clinical isolate. A recombinant plasmid, pAST-1, expressed the beta-lactamase AST-1 in Escherichia coli JM109. Its pI was 4.8, and its relative molecular mass was 31 kDa. E. coli JM109(pAST-1) was resistant to penicillins and narrow-spectrum cephalosporins. The beta-lactamase AST-1 had a restricted hydrolytic activity spectrum. Its activity was partially inhibited by clavulanic acid but not by sulbactam and tazobactam. AST-1 is an Ambler class A beta-lactamase sharing 65% amino acid identity with beta-lactamase FAR-1, the most closely related enzyme.     

Variability of chromosomally encoded beta-lactamases from Klebsiella oxytoca.

The beta-lactamase genes of Klebsiella oxytoca were previously divided into two main groups: bla(OXY-1) and bla(OXY-2). The two beta-lactamase groups were each represented by beta-lactamases with four different pIs. In each group, one form of beta-lactamase is more frequent than the others combined. The beta-lactamase gene of each representative beta-lactamase with a different pI that was not yet sequenced (pIs 5.7, 6.8 [OXY-2], 7.1, 8.2, and 8.8 [OXY-1]) was cloned and sequenced. The susceptibility patterns as well as relative rates and kinetic parameters for beta-lactam hydrolysis revealed that OXY-2 enzymes hydrolyzed several of the beta-lactams that were examined (carbenicillin, cephalothin, cefamandole, ceftriaxone, and aztreonam) at a greater rate than the OXY-1 enzymes did. Comparison of K. oxytoca beta-lactamases with plasmid-mediated extended-spectrum beta-lactamases MEN-1 and TOHO-1 implied that the threonine at position 168 present in OXY-2 beta-lactamase instead of the alanine in OXY-1 could be responsible for its modified substrate hydrolysis. In each group, the beta-lactamase with a variant pI differs from the main form of beta-lactamase by one to five amino acid substitutions. The substrate profile and the 50% inhibitory concentrations revealed that all substitutions differing from the main form of beta-lactamase were neutral except one difference in the OXY-1 group. This substitution of an Ala to a Gly at position 237 increases the hydrolysis of some beta-lactams, particularly aztreonam; decreases the hydrolysis of benzylpenicillin, cephaloridine, and cefamandole, and decreases the susceptibility to clavulanic acid (fivefold increase in the 50% inhibitory concentration).     

Sequence and genome context analysis of a new molecular class D beta-lactamase gene from Legionella pneumophila

Legionella pneumophila Philadelphia-1 (ATCC 33152) produces a serine active site beta-lactamase. The chromosomal gene that encodes this enzyme, loxA, has been cloned by PCR using information from the L. pneumophila Philadelphia-1 genome sequencing project. LoxA is a class 2d penicillinase, and its sequence puts it into the molecular class D beta-lactamase family, although phylogenetic analysis shows that LoxA forms a distinct branch in the OXA family along with the LoxA homologue, OXA-29, from Legionella gormanii ATCC 33297(T). Upstream of loxA on the L. pneumophila Philadelphia-1 chromosome is a two-gene locus similar to that found linked to the beta-lactamase genes of Gram-positive bacteria. The unit consists of loxI, encoding a homologue of the Gram-positive beta-lactamase expression regulator, and pbpX, encoding a putative penicillin-binding transpeptidase. Despite the presence of beta-lactamase regulator homologues, we could find no evidence of LoxA induction upon challenge of L. pneumophila Philadelphia-1 with beta-lactams.     

Genetic and biochemical characterization of CGB-1, an Ambler class B carbapenem-hydrolyzing beta-lactamase from Chryseobacterium gleum

Chryseobacterium gleum (previously included in the Flavobacterium IIb species) is a gram-negative aerobe that is a source of nosocomial infections. An Ambler class B beta-lactamase gene was cloned and expressed in Escherichia coli from reference strain C. gleum CIP 103039 that had reduced susceptibility to expanded-spectrum cephalosporins and carbapenems. The purified beta-lactamase, CGB-1, with a pI value of 8.6 and a determined relative molecular mass of ca. 26 kDa, hydrolyzed penicillins; narrow- and expanded-spectrum cephalosporins; and carbapenems. CGB-1 was a novel member of the molecular subclass B1 of metallo-enzymes. It had 83 and 42% amino acid identity with IND-1 from Chryseobacterium indologenes and BlaB from C. meningosepticum, respectively. Thus, in addition to the previously characterized clavulanic acid-inhibited extended-spectrum beta-lactamase CGA-1 of Ambler class A, C. gleum produces a very likely chromosome-borne class B beta-lactamase.     

Genes involved in the regulation of beta-lactamase production in enterococci and staphylococci.

In enterococci, the structural gene for beta-lactamase (blaZ) is identical to blaZ from Staphylococcus aureus. However, in the enterococci studied to date, beta-lactamase is produced constitutively, whereas in staphylococci it is often inducible. Recent reports have revealed the presence of two adjacent genes upstream of the staphylococcal blaZ thought to be the antirepressor (blaR1) and repressor (blaI) genes. In the present study, beta-lactamase expression mutants of the staphylococcal beta-lactamase plasmid pI524 were generated by transposon mutagenesis with the transposon Tn917. Tn917 insertions upstream of blaZ in either blaR1 or blaI resulted in constitutive beta-lactamase production, indicating that the repressor function is lost with insertion of Tn917 into either gene. This finding supports the concept that the staphylococcal beta-lactamase regulatory genes are encoded on a polycistronic mRNA. The corresponding region upstream of the enterococcal blaZ from Enterococcus faecalis HH22 was sequenced and compared with the staphylococcal blaR1 sequence. The two sequences were identical for 893 nucleotides, and then the sequences diverged completely. Therefore, in strain HH22, only 51% of the putative antirepressor gene is present and the repressor gene is also absent. In conclusion, constitutive beta-lactamase production in HH22 appears to be due to a lack of the regulatory genes blaR1 and blaI which regulate expression of blaZ in staphylococci.     

blaI and blaR1 regulate beta-lactamase and PBP 2a production in methicillin-resistant Staphylococcus aureus.

For Staphylococcus aureus, it is hypothesized that two genes located upstream of the beta-lactamase gene, blaZ, are required for the inducible expression of beta-lactamase. blaR1 is predicted to encode a signal-transducing membrane protein, and blaI is predicted to encode a repressor protein. These same two genes may also regulate the production of penicillin-binding protein 2a (PBP 2a), a protein essential for expression of methicillin resistance. To confirm that these two genes encode products that can control both beta-lactamase and PBP 2a production, blaI, blaR1, and blaZ with a 150-nucleotide deletion at the 3' end were subcloned from a 30-kb staphylococcal beta-lactamase plasmid and three beta-lactamase-negative strains of methicillin-resistant S. aureus were transformed with the recombinant plasmid containing that insert. The production of PBP 2a and a nonfunctional beta-lactamase was detected by fluorography and by immunoblots with polyclonal antisera directed against each of the proteins. Whereas the parent strains did not produce beta-lactamase and constitutively produced PBP 2a, PBP 2a and a truncated beta-lactamase were now inducible in the transformants. Therefore, two plasmid-derived genes regulate the production of both PBP 2a and beta-lactamase.     

Binding properties of a peptide derived from beta-lactamase inhibitory protein.

To overcome the antibiotic resistance mechanism mediated by beta-lactamases, small-molecule beta-lactamase inhibitors, such as clavulanic acid, have been used. This approach, however, has applied selective pressure for mutations that result in beta-lactamases no longer sensitive to beta-lactamase inhibitors. On the basis of the structure of beta-lactamase inhibitor protein (BLIP), novel peptide inhibitors of beta-lactamase have been constructed. BLIP is a 165-amino-acid protein that is a potent inhibitor of TEM-1 beta-lactamase (K(i) = 0.3 nM). The cocrystal structure of TEM-1 beta-lactamase and BLIP indicates that residues 46 to 51 of BLIP make critical interactions with the active site of TEM-1 beta-lactamase. A peptide containing this six-residue region of BLIP was found to retain sufficient binding energy to interact with TEM-1 beta-lactamase. Inhibition assays with the BLIP peptide reveal that, in addition to inhibiting TEM-1 beta-lactamase, the peptide also inhibits a class A beta-lactamase and a class C beta-lactamase that are not inhibited by BLIP. The crystal structures of class A and C beta-lactamases and two penicillin-binding proteins (PBPs) reveal that the enzymes have similar three-dimensional structures in the vicinity of the active site. This similarity suggests that the BLIP peptide inhibitor may have a broad range of activity that can be used to develop novel small-molecule inhibitors of various classes of beta-lactamases and PBPs.     

Monitoring beta-lactamase activity in vivo by 13C nuclear magnetic resonance spectroscopy

A 13C-labeled cephalothin, 7 beta-(2-thienylacetamido)-3-[acetoxy-13C1]methyl-3-cephem-4- carboxylate (compound 1), has been prepared and used to monitor beta-lactamase activities by 13C nuclear magnetic resonance spectroscopy. Time-elapsed spectral analysis of the reaction of the labeled cephalothin with the TEM-2 beta-lactamase purified from Escherichia coli revealed the progressive loss of the cephalothin acetyl resonance at 176.8 ppm and accumulation of an acetate signal at 184.3 ppm. Spectral results identical to those observed in the in vitro experiment were obtained when compound 1 was incubated with cell suspensions of E. coli JSR-O (pBR322), which contains the plasmid-encoded TEM-2 beta-lactamase, and Enterobacter cloacae strains that contain a class I chromosomal beta-lactamase. Pseudo-first-order rate constants for the lactamase-catalyzed formation of acetate from cephalothin in vivo were obtained by integration of the 13C-acetyl resonances of compound 1 during timed incubations with cell preparations. These results constitute the first demonstration of the ability to monitor beta-lactamase activity in viable cells by nuclear magnetic resonance spectroscopy.     

Carbapenem resistance in Escherichia coli associated with plasmid-determined CMY-4 beta-lactamase production and loss of an outer membrane protein

Three cefoxitin-resistant Escherichia coli isolates from stool specimens of a patient with leukemia were either resistant, intermediate, or sensitive to imipenem. Conjugation experiments showed that cefoxitin resistance, but not imipenem resistance, was transferable. All isolates were shown by isoelectric focusing to produce two beta-lactamases with isoelectric points of 5.4 (TEM-1, confirmed by sequencing of a PCR product) and >8.5 (consistent with a class C beta-lactamase). The gene coding for the unknown beta-lactamase was cloned and sequenced and revealed an enzyme which had 99.9% sequence identity with the plasmid-determined class C beta-lactamase CMY-2. The cloned beta-lactamase gene differed from blaCMY-2 at one nucleotide position that resulted in an amino acid change, tryptophan to arginine at position 221. We propose that this enzyme be designated CMY-4. Both the imipenem-resistant and -intermediate isolates lacked a 38-kDa outer membrane protein (OMP) that was present in the imipenem-sensitive isolate. The lack of an OMP alone did not explain the difference in carbapenem susceptibilities observed. However, measurement of beta-lactamase activities (including measurements under conditions where TEM-1 beta-lactamase was inhibited) indicated that the imipenem-intermediate isolate expressed six- to eightfold less beta-lactamase than did the other isolates. This study illustrates that carbapenem resistance in E. coli can arise from high-level expression of plasmid-mediated class C beta-lactamase combined with an OMP deficiency. Furthermore, in the presence of an OMP deficiency, the level of expression of a plasmid-mediated class C beta-lactamase is an important factor in determining whether E. coli isolates are fully resistant to carbapenems.     

Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the foss of an outer membrane protein.

Six Escherichia coli and 12 Klebsiella pneumoniae isolates from a single hospital expressed a common beta-lactamase with a pI of approximately 9.0 and were resistant to cefoxitin and cefotetan (MIC ranges, 64 to > 128 and 16 to > 128 micrograms/ml, respectively). Seventeen of the 18 strains produced multiple beta-lactamases. Most significantly, three K. pneumoniae strains were also resistant to imipenem (MICs, 8 to 32 micrograms/ml). Spectrophotometric beta-lactamase assays with purified enzyme indicated hydrolysis of cephamycins, in addition to cephaloridine and benzylpenicillin. The 4ene encoding the pI 9.0 beta-lactamase (designated ACT-1 for AmpC type) was cloned and sequenced, which revealed an ampC-type beta-lactamase gene that originated from Enterobacter cloacae and that had 86% sequence homology to the P99 beta-lactamase and 94% homology to the partial sequence of MIR-1. Southern blotting revealed that the gene encoding ACT-1 was on a large plasmid in some of the K. pneumoniae strains as well as on the chromosomes of all of the strains, suggesting that the gene is located on an easily mobilized element. Outer membrane protein profiles of the K. pneumoniae strains revealed that the three imipenem-resistant strains were lacking a major outer membrane protein of approximately 42 kDa which was present in the imipenem-susceptible strains. ACT-1 is the first plasmid-mediated AmpC-type beta-lactamase derived from Enterobacter which has been completely sequenced. This work demonstrates that in addition to resistance to cephamycins, imipenem resistance can occur in K. pneumoniae when a high level of the ACT-1 beta-lactamase is produced in combination with the loss of a major outer membrane protein.     

Genetically diverse ceftazidime-resistant isolates from a single center: biochemical and genetic characterization of TEM-10 beta-lactamases encoded by different nucleotide sequences.

Ceftazidime-resistant isolates of Escherichia coli and Klebsiella pneumoniae produced a plasmid-mediated beta-lactamase with a pI of 5.6 with biochemical characteristics comparable to those of the TEM-10 beta-lactamase. Plasmids from the two strains were nonidentical. Both TEM-10 sequences differed from TEM-1 by substitutions of Ser-162 and Lys-237. The nucleotide sequences of the two genes were identical except for three silent nucleotide substitutions corresponding to the nucleotide differences in the Tn2 TEM-1 or Tn3 TEM-1 genes. The original TEM-10 plasmid was identical to that found in the E. coli isolate and coded for a gene that corresponded to the TEM-10 beta-lactamase from Tn2.     

Sequences of the NPS-1 and TLE-1 beta-lactamase genes

The NPS-1 and TLE-1 beta-lactamase genes were cloned and sequenced. NPS-1 differed from LCR-1 beta-lactamase in 8 of 260 amino acids. TLE-1 differed from TEM-1 by a single Asp(115)-->Gly substitution and has been renamed TEM-90.     

Antibodies against chromosomal beta-lactamase.

A murine monoclonal anti-chromosomal beta-lactamase antibody was developed and an immunoblotting technique was used to study the presence of serum and sputum antibodies against Pseudomonas aeruginosa chromosomal group 1 beta-lactamase in patients with cystic fibrosis (CF). The serum antibody response was studied with serum samples collected in 1992 from 56 CF patients in a cross-sectional study and with serum samples from 18 CF patients in a longitudinal study. Anti-beta-lactamase immunoglobulin G antibodies were present in all of the serum samples from the patients with chronic bronchopulmonary P. aeruginosa infection (CF + P) but in none of the CF patients with no or intermittent P. aeruginosa infection. Anti-beta-lactamase antibodies were present in serum from CF + P patients after six antipseudomonal courses (median) and correlated with infection with a beta-lactam-resistant strain of P. aeruginosa. The sputum antibody response and the beta-lactamase activity in sputum samples from 14 of the CF + P patients were also studied. beta-lactamase antibodies were present in 10 of these samples. P. aeruginosa strains isolated from these samples were partially derepressed, producing group 1 cephalosporinase. We found a wide range of chromosomal beta-lactamase activity in the sputum samples, with no correlation with basal or induced activity of beta-lactamase expression. The presence of anti-beta-lactamase antibodies in endobronchial sputum could be an important factor in the defense against the infection. On the other hand, immune complexes between the beta-lactamase and corresponding antibodies could play a role in the pathogenesis of bronchopulmonary injury in CF by mediating hyperimmune reactions.     

Reevaluation of the factors involved in the efficacy of new beta-lactams against Enterobacter cloacae.

The roles of outer membrane permeability, beta-lactamase stability, and inhibition of penicillin-binding proteins in the activity of new beta-lactams against Enterobacter cloacae were reappraised by using several methodological improvements. Outer membrane permeability in intact cells was determined by using a high-pressure liquid chromatography (HPLC)-based technique that avoided certain possible artifacts of the traditional methods. Vmax values were calculated from the numbers of enzyme molecules produced per cell and from catalytic constant (Kcat) values, which were obtained with purified beta-lactamase. Minimal periplasmic antibiotic concentrations needed to inhibit bacterial cell wall synthesis were estimated from the Zimmermann-Rosselet equation. All the beta-lactams tested formed relatively stable complexes with purified beta-lactamase. The antibiotics that exhibited low affinity for beta-lactamase apparently needed higher periplasmic concentrations to inhibit cell wall synthesis, suggesting a possible correlation between the affinity of beta-lactamase and the affinity of penicillin-binding proteins for the new beta-lactams. By using these estimates of outer membrane permeability, beta-lactamase hydrolysis, and cell wall-inhibiting concentrations, MIC could be theoretically predicted to within 1 dilution for five beta-lactams in three isogenic E. cloacae strains with differences in antibiotic susceptibility due to different porin or beta-lactamase contents.     

TLA-1: a new plasmid-mediated extended-spectrum beta-lactamase from Escherichia coli

Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids that encoded two beta-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from E. coli R170 to E. coli J53-2. The transferred plasmid, RZA92, which encoded a single beta-lactamase, was 150 kb in length. The cefotaxime resistance gene that encodes the TLA-1 beta-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E. coli DH5alpha. Sequencing of the bla(TLA-1) gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A beta-lactamases: (70)SXXK, (130)SDN, and (234)KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A beta-lactamase from Chryseobacterium (Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A beta-lactamase from E. coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 of Salmonella typhimurium; and 39% identity with CepA of Bacteroides fragilis. The partially purified TLA-1 beta-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum beta-lactamase of Ambler class A.     

Molecular and biochemical characterization of a carbapenem-hydrolysing beta-lactamase from Flavobacterium johnsoniae

Flavobacterium johnsoniae CIP100931 is resistant to most beta-lactam antibiotics and has a decreased susceptibility to carbapenems. A beta-lactamase gene was cloned and expressed in Escherichia coli DH10B. The purified beta-lactamase, JOHN-1, with a pI value of 9.0 and with a determined relative molecular mass of approximately 27 kDa was found to be a monomeric zinc-dependent enzyme that hydrolyses penicillins, narrow- and expanded-spectrum cephalosporins, carbapenems, but not monobactams. Sequence analysis revealed that JOHN-1 is a molecular class B beta-lactamase that is most closely related to BlaB from Chryseobacterium meningosepticum and IND-1 from Chryseobacterium indologenes (47% and 41% amino acid identity, respectively). JOHN-1 is a new member of the highly divergent subclass B1 lineage of metallo-enzymes. Although F. johnsoniae and Chryseobacterium spp. are phylogenetically related bacteria, this report further underlines the heterogeneity of class B beta-lactamases that are naturally produced by environmental Gram-negative aerobes and that are now recognized as the most important reservoir for these beta-lactamase genes.     

Mutants of the TEM-1 beta-lactamase conferring resistance to ceftazidime

Spontaneous ceftazidime resistant mutants were obtained from an Escherichia coli K12 J62-2 expressing the TEM-1 beta-lactamase (mutation frequency = 10(-9). These mutants produced beta-lactamases with similar molecular weights, kinetic parameters and isoelectric points (pI) to the beta-lactamases produced by ceftazidime resistant clinical isolates which have recently been identified in this laboratory. Mutant enzyme A focused as a doublet band at pI 5.3 with an additional weak pI 5.4 band. The doublet co-focused with the TEM-E2 beta-lactamase, produced by a ceftazidime resistant Klebsiella oxytoca isolate, which was originally obtained in a Liverpool hospital. Mutant enzyme B had a pI identical to the TEM-E1 beta-lactamase produced by a ceftazidime resistant clinical isolate of E. coli found in Belgium. These results suggest that the two beta-lactamases in the clinical strains may have come from simple mutations of the TEM-1 beta-lactamase gene.     

Epidemiology of conjugative plasmid-mediated AmpC beta-lactamases in the United States

A sample of 752 resistant Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli strains from 70 sites in 25 U.S. states and the District of Columbia was examined for transmissibility of resistance to ceftazidime and the nature of the plasmid-mediated beta-lactamase involved. Fifty-nine percent of the K. pneumoniae, 24% of the K. oxytoca, and 44% of the E. coli isolates transferred resistance to ceftazidime. Plasmids encoding AmpC-type beta-lactamase were found in 8.5% of the K. pneumoniae samples, 6.9% of the K. oxytoca samples, and 4% of the E. coli samples, at 20 of the 70 sites and in 10 of the 25 states. ACT-1 beta-lactamase was found at eight sites, four of which were near New York City, where the ACT-1 enzyme was first discovered; ACT-1 beta-lactamase was also found in Massachusetts, Pennsylvania, and Virginia. FOX-5 beta-lactamase was also found at eight sites, mainly in southeastern states but also in New York. Two E. coli strains produced CMY-2, and one K. pneumoniae strain produced DHA-1 beta-lactamase. Pulsed-field gel electrophoresis and plasmid analysis suggested that AmpC-mediated resistance spread both by strain and plasmid dissemination. All AmpC beta-lactamase-containing isolates were resistant to cefoxitin, but so were 11% of strains containing transmissible SHV- and TEM-type extended-spectrum beta-lactamases. A beta-lactamase inhibitor test was helpful in distinguishing the two types of resistance but was not definitive since 24% of clinical isolates producing AmpC beta-lactamase had a positive response to clavulanic acid. Coexistence of AmpC and extended-spectrum beta-lactamases was the main reason for these discrepancies. Plasmid-mediated AmpC-type enzymes are thus responsible for an appreciable fraction of resistance in clinical isolates of Klebsiella spp. and E. coli, are disseminated around the United States, and are not so easily distinguished from other enzymes that mediate resistance to oxyimino-beta-lactams.     

Resistance to beta-lactams in Mycobacterium fortuitum.

It is widely assumed that the high level of intrinsic resistance to beta-lactam antibiotics exhibited by mycobacteria results from the combination of factors including permeability to the drugs, beta-lactamase production, and affinity for penicillin-binding proteins (PBPs). We conducted an evaluation of the second and third factors by isolating nitrosoguanidine-induced mutants from the beta-lactamase-producing strain Mycobacterium fortuitum ATCC 19542 that displayed either elevated or reduced resistance to various beta-lactam antibiotics. The mutants studied included D1 (a beta-lactamase producer with high penicillin resistance), gamma 27 (a low-level beta-lactamase producer with low penicillin resistance), and D316 (a high-level beta-lactamase producer with high penicillin resistance). In all strains examined, four major PBPs, named 1, 2a, 2b, and 3, with apparent molecular weights of 102,000, 90,000, 87,000, and 50,000, respectively, were found. The MICs of various beta-lactams toward ATCC 19542 and its mutants were considered in the context of beta-lactamase production, the quantity of PBPs synthesized, and their affinities for beta-lactam antibiotics. The data obtained show that beta-lactamase production is likely to be an important factor in the expression of resistance by clinical isolates and that PBP alterations can contribute to resistance at least in laboratory-derived mutants.