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ACC-4, an omega loop mutant (Val(211)-->Gly) of the Hafnia alvei-derived cephalosporinase ACC-1, was encoded by an Escherichia coli plasmid. The genetic environment of bla(ACC-4) shared similarities with plasmidic regions carrying bla(ACC-1). Kinetics of beta-lactam hydrolysis and levels of resistance to beta-lactams showed that ACC-4 was more effective than ACC-1 against expanded-spectrum cephalosporins.

A strain of Klebsiella pneumoniae resistant to cefoxitin and oxyimino-cephalosporins, but susceptible to cefepime, was isolated from an adult patient hospitalised in Taichung, Taiwan. Isoelectric focusing revealed three beta-lactamases with isoelectric points of 5.4, 8.2 and 7.9, respectively. Following PCR with plasmid DNA templates and gene sequencing, these enzymes were shown to correspond to TEM-1, SHV-5 and a novel DHA-1-like enzyme (designated DHA-3). The bla genes for TEM-1 and SHV-5 were transferable, but the bla(DHA-3) gene was non-self-transferable in conjugation experiments. All three bla genes were successfully introduced by electrotransformation into an Escherichia coli recipient (DH5alpha), resulting in a similar resistance profile to that observed in the original donor strain. Other K. pneumoniae strains producing DHA-1-like enzymes have been identified previously in Taiwan, and this report suggests that DHA-type beta-lactamases are continuing to emerge in this country.

Shotgun cloning experiments with restriction enzyme-digested genomic DNA from Morganella morganii 1, which expresses high levels of cephalosporinase, into the pBKCMV cloning vector gave a recombinant plasmid, pPON-1, which encoded four entire genes: ampC, ampR, an hybF family gene, and orf-1 of unknown function. The deduced AmpC beta-lactamase of pI 7.6 shared structural and functional homologies with AmpC from Citrobacter freundii, Escherichia coli, Yersinia enterocolitica, Enterobacter cloacae, and Serratia marcescens. The overlapping promoter organization of ampC and ampR, although much shorter in M. morganii than in the other enterobacterial species, suggested similar AmpR regulatory properties. The MICs of beta-lactams for E. coli MC4100 (ampC mutant) harboring recombinant plasmid pACYC184 containing either ampC and ampR (pAC-1) or ampC (pAC-2) and induction experiments showed that the ampC gene of M. morganii 1 was repressed in the presence of ampR and was activated when a beta-lactam inducer was added. Moreover, transformation of M. morganii 1 or of E. coli JRG582 (delta ampDE) harboring ampC and ampR with a recombinant plasmid containing ampD from E. cloacae resulted in a decrease in the beta-lactam MICs and an inducible phenotype for M. morganii 1, thus underlining the role of an AmpD-like protein in the regulation of the M. morganii cephalosporinase. Fifteen other M. morganii clinical isolates with phenotypes of either low-level inducible cephalosporinase expression or high-level constitutive cephalosporinase expression harbored the same ampC-ampR organization, with the hybF and orf-1 genes surrounding them; the organization of these genes thus differed from those of ampC-ampR genes in C. freundii and E. cloacae, which are located downstream from the fumarate operon. Finally, an identical AmpC beta-lactamase (DHA-1) was recently identified as being plasmid encoded in Salmonella enteritidis, and this is confirmatory evidence of a chromosomal origin of the plasmid-mediated cephalosporinases.

Bacteroides fragilis CS30 is a clinical isolate resistant to high concentrations of benzylpenicillin and cephaloridine but not to cephamycin or penem antibiotics. beta-Lactam resistance is mediated by a chromosomally encoded cephalosporinase produced at a high level. The gene encoding this beta-lactamase was cloned from genomic libraries constructed in Escherichia coli and then mated with B. fragilis 638 for identification of ampicillin-resistant (Apr) strains. Apr transconjugants contained a nitrocefin-reactive protein with the physical and enzymatic properties of the original CS30 isolate. The beta-lactamase gene (cepA) was localized by deletion analysis and subcloned, and its nucleotide sequence was determined. The 903-bp cepA open reading frame encoded a 300-amino-acid precursor protein (predicted molecular mass, 34,070 Da). A beta-lactamase-deficient mutant strain of B. fragilis 638 was constructed by insertional inactivation with the cepA gene of CS30, demonstrating strict functional homology between these chromosomal beta-lactamase genes. An extensive comparison of the CepA protein sequence by alignment with other beta-lactamases revealed the strict conservation of at least four elements common to Ambler class A. A further comparison of the CepA protein sequence with protein sequences of beta-lactamases from two other Bacteroides species indicated that they constitute their own distinct subgroup of class A beta-lactamases.

A clinical isolate of Bacteroides vulgatus was resistant to tetracycline, clindamycin, ampicillin, cephaloridine, cefoxitin, and other beta-lactam antibiotics except imipenem. beta-Lactam resistance was mediated by a membrane-associated, clavulanate-sensitive cephalosporinase capable of degrading cephalosporins and penicillins. Cefoxitin also was degraded but at a slow rate. The cefoxitin resistance (Fxr) determinant was cloned from B. vulgatus genomic libraries that were prepared in Escherichia coli and then mated with Bacteroides fragilis for the identification of Fxr strains. Analysis of B. fragilis strains with the cloned Fxr determinant revealed the presence of a new beta-lactamase protein with the physical and enzymatic properties of the beta-lactamase found in the original B. vulgatus isolate. The beta-lactamase gene (cfxA) was subcloned on a 2.2-kb DraI-HindIII fragment, and the nucleotide sequence was determined. These results showed that cfxA encoded a protein of 321 amino acids and 35,375 molecular weight. Mutant strains in which the cfxA structural gene was disrupted by insertional inactivation lost both Fxr and beta-lactamase activity. Comparison of CfxA with other beta-lactamases showed a relationship with the active-site serine beta-lactamases in the Ambler molecular class A, although CfxA had apparently diverged significantly. This was exemplified by the substitution in CfxA at 13 of 25 amino acid residues previously identified as being invariant in class A beta-lactamases. These results suggest that CfxA may represent a new class A homology group which diverged very early.

A beta-lactamase was purified 430-fold from the culture supernatant of Acinetobacter calcoaceticus by ion exchange chromatography on CM-Sephadex and affinity chromatography on phenylboronic-acid-agarose. The purified enzyme was homogeneous as judged by SDS-PAGE, and was characterized with respect to molecular mass (38 and 41 kDa by gel filtration on Sephadex G-75 and SDS-PAGE, respectively), pH optimum (pH 7.0), temperature optimum (45 degrees C) and isoelectric point (9.3). The beta-lactamase showed mainly cephalosporinase activity. It was inhibited by cloxacillin, carbenicillin, penicillanic acid sulphone (sulbactam) and aztreonam. It was not inhibited by clavulanic acid up to a concentration of 0.25 mM. Neither EDTA nor p-chlormercuribenzoate, up to concentrations of 1 or 100 mM, respectively, affected activity. According to these characteristics, it is a typical CEP-N cephalosporinase.

A new plasmid-mediated beta-lactamase (FPM-1) with an isoelectric point of 7.2 and a molecular weight of 26,000 was found in a cefuroxime-resistant clinical isolate of Proteus mirabilis strain 6003. FPM-1 can be classified as a type I oxyimino-cephalosporinase on the basis of its substrate specificity and inhibition pattern by clavulanic acid etc., and its conferred resistance on both the strain and transconjugants against most oxyme-type cephalosporins as well as the older ones but not against cefamycins and a few exceptional oxyme-type cephalosporins such as ceftizoxime, ceftazidime and cefixime. In a murine systemic infection model, only these FPM-1-stable drugs exhibited protective activity against the FPM-1-producing P. mirabilis 6003 similar to that against a nonproducing derivative strain. The FPM-1-mediated cefuroxime resistance in P. mirabilis 6003 was transferred to co-infected Escherichia coli 7004 at frequencies between 3.8 x 10(-3) and 4.0 x 10(-2) in a murine ascending urinary tract infection model. In the same infection model due to the FPM-1-producing E. coli transconjugant, FPM-1-stable cefixime was significantly more effective than FPM-1-labile cefteram pivoxil, although both drugs had similar therapeutic effect against its FPM-1-nonproducing counterpart strain.

The cephalosporinase of Citrobacter freundii GN346 is a class C beta-lactamase, consisting of 361 amino acids and exhibiting the substrate profile of a typical cephalosporinase. On the conversion of a conserved glutamic acid at residue 219 to lysine, the substrate spectrum of the cephalosporinase was extended to oxyimino cephalosporins, aztreonam and carbenicillin, which are essentially undesirable substrates for the enzyme. Escherichia coli cells carrying the mutant gene showed higher resistance levels to cefuroxime, aztreonam, and carbenicillin, but a lower resistance level to cefoxitin, than cells carrying the wild gene. The kcat values of the purified mutant enzyme for ceftazidime, cefuroxime, and cefmenoxime were 77,100, and 300 times those of the wild enzyme, respectively. The relative Vmax values of the mutant enzyme for aztreonam and carbenicillin were determined to be 11 and 23 times those of the wild enzyme, respectively, but the value of the mutant enzyme for cefoxitin was only one-third that of the wild enzyme.

Citrobacter freundii GN346 produces a class C beta-lactamase exhibiting the substrate profile of a typical cephalosporinase. The structural and promoter regions of the cephalosporinase gene, comprising 1408 nucleotides, were completely sequenced. The amino acid sequence of the mature enzyme, comprising 361 amino acids, and its molecular mass, 39,878 Da, were determined. The active site was confirmed to be Ser-64. The amino acid sequence of the enzyme differs from that of the cephalosporinase of C. freundii OS60 by nine residues. The nucleotide sequence of the promoter region suggests a possible attenuator structure. Lys-67, one of the most conserved residues found in class A and C beta-lactamases and penicillin-binding proteins, was converted into arginine, threonine or glutamic acid through site-directed mutagenesis. The Glu-67 enzyme had lost the catalytic activity and the Thr-67 enzyme only showed a trace of activity. The Arg-67 enzyme, which retained a significant amount of the activity, was purified. The Km values of the Arg-67 enzyme for cephalothin, cephaloridine and benzylpenicillin are 13-19 times those of the wild-type enzyme; the kcat values for the three substrates are 37%, 3%, and 36% those of the wild-type enzyme, respectively.

Cephalosporin beta-lactamase (cephalosporinase) was purified from a strain of Escherichia coli resistant to beta-lactam antibiotics. The purified enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis, and its molecular weight was 39,000 from sodium dodecyl sulfate-acrylamide gel electrophoresis. Its isoelectric point was 8.7. The specific activity was 31.7 mumol/min per mg of protein of the purified enzyme for the hydrolysis of cephaloridine. The optimal pH was about 8.0, and the optimal temperature was 36 degrees C. The enzyme activity was inhibited by iodine, some divalent metallic ions, semisynthetic penicillins, cefuroxime-type cephalosporins, and cephamycin derivatives. The enzymological properties of the purified preparation have been compared with those of beta-lactamases from other gram-negative bacteria.

A hospital strain of Enterobacter aerogenes (MULB 250) isolated from a urinary tract infection was found to be cephalosporin and ampicillin resistant and carbenicillin susceptible. The beta-lactamase produced by this strain was extracted and purified by means of affinity chromatography, using a cephalosporin C-bound Sepharose 4B column. The purified enzyme was tested for hydrolysis of penicillin and various cephalosporins. The K(m) value is 11.8 muM for benzyl penicillin and 130 muM for cephalosporin C. The isoelectric point of the enzyme is 9.3, and its molecular weight is 29,500 +/- 1,000. Rabbit antiserum obtained against this MULB 250 beta-lactamase showed no cross-reaction with other penicillinases or cephalosporinases in neutralization tests. Comparisons of results obtained with other beta-lactamases, particularly from Enterobacter cloacae P99, indicate that the Enterobacter MULB 250 enzyme presents a typical cephalosporinase profile. As far as we know, this type of enzyme is relatively rare.

A cephalosporin beta-lactamase (cephalosporinase) was extracted from Enterobacter cloacae GN7471 and purified by means of column chromatography. The resulting preparation gave a single protein band upon polyacrylamide gel electrophoresis. The enzyme's isoelectric point was 8.4, and its molecular weight was 44,000. The optimal pH was 8.5, and the optimal temperature was 40 degrees C. The enzyme hydrolyzed cephalosporins much more readily than penicillins. The enzyme activity was inhibited by iodine, semisynthetic penicillins, cefuroxime-type cephalosporins, and cephamycin derivatives. The enzymological properties of the purified enzyme were compared with those of beta-lactamases derived from other gram-negative enteric bacteria.

1. Mutants of Bacillus cereus 569/H/9 have been screened in a search for strains that synthesize variants of beta-lactamase II. 2. One of these mutants (strain 569/H/9/1) produces a beta-lactamase II-like enzyme that shows a selective decrease in cephalosporinase activity. 3. beta-Lactamase II from strain 569/H/9/1 has been purified to apparent homogeneity and its kinetic properties have been examined. This enzyme resembles the parent beta-lactamase II in its relative activity with benzylpenicillin as substrate when Zn(II) is replaced by other metal ions, but differs detectably from the parent enzyme in its isoelectric point.

The inhibition of Citrobacter freundii cephalosporinase activity by moxalactam is shown to be due to the formation of a transiently stable covalent complex, probably acyl enzyme. The covalent complex formed was identified by coelution of [14C] moxalactam with the enzyme by using Sephadex G-25 gel filtration in the presence of 5.7 M guanidine hydrochloride and by analytical isoelectric focusing. Both the side-chain carboxyl group and the 7 alpha-methoxy group of moxalactam were necessary to stabilize the complex. Moxalactam is racemic with respect to the alpha carbon of the 7 beta-acylamino side chain, and the complex with the R epimer (half-life, 4.6 min) decomposed much more rapidly than that formed with the S epimer (half-life, 130 min). For other beta-lactam antibiotics that were stable to beta-lactamase, the half-lives of enzyme-antibiotic complexes were less than 4 min.

An inducible cephalosporinase was purified from Pseudomonas maltophilia GN12873. The pI was 8.4, and the molecular weight was ca. 56,000 by gel filtration or 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that this enzyme had two subunits. The optimal pH and optimal temperature were 7.5 and 45 degrees C, respectively. Enzyme activity was inhibited by clavulanic acid, sulbactam, cephamycin derivatives, carbapenem antibiotics, iodine, HgCl2, and p-chloromercuribenzoate. The enzyme showed a broad substrate profile, hydrolyzing cephaloridine, cefazolin, cefsulodin, penicillin G, ceftizoxime, and ampicillin at a high rate.

Both a penicillinase and a cephalosporinase are present in a strain of Citrobacter freundii (GN7391) resistant to beta-lactam antibiotics. The penicillinase was identical to the type Ia penicillinases (Type III by Richmond classification), mediated by Rms212 and R-TEM. A cephalosporinase, typical of enterobacteriaceae chromosomal beta-lactamase (Type I by Richmond classification), was purified from the strain. It gave a single protein band on polyacrylamide gel electrophoresis and immunoelectrophoresis; the pI was 8.6 and its molecular weight was approximately 38 000. Cysteine was not found among its amino acids. The specific activity was 388 units (mg protein)-1 for the hydrolysis of cephaloridine, and the optimal pH was 8.0. Rabbit antiserum obtained against the purified enzyme showed cross-reaction with cephalosporinases produced by strains of Enterobacter cloacae in a neutralization test.

A mutant of Bacillus cereus 5/B, strain 5/B/6, produces a beta-lactamase II-like enzyme but no beta-lactamase I. Beta-lactamases II and II 5/B/6 appear to show a high degree of homology, but there are significant differences in their enzymic properties.

The properties of cefazolin, cefoxitin, cefamandole, and cefuroxime have been compared with those of cephaloridine, cephalothin, and cephalexin. The properties included for examination are the susceptibility to a range of beta-lactamases commonly encountered in gram-negative species and the ability of a beta-lactam antibiotic to penetrate the outer layers of Escherichia coli. Of the antibiotics tested, cefamandole was the most active in its antibiotic activity but had the disadvantage that it was sensitive to hydrolysis by type IVc beta-lactamase. Cefoxitin and cefuroxime were slightly less active than cefamandole but were effectively resistant to all the beta-lactamases used in the test.

BETA-Lactamases (EC. 3.5.2.6) from strains of Gram-negative bacteria have been studied using analytical isoelectric focusing. This permits a visual comparison of the patterns of beta-lactamase bands produced by enzymes from different organisms. Purification of crude intracellular preparations is unnecessary and the technique is sufficiently sensitive to demonstrate beta-lactamase in mutants previously reported to lack the enzyme. R that have not been distinguished from one another biochemically or immunologically can be differentiated by isoelectric focusing. Conversely, the enzymes specified by the R factors RTEM, R1 and RGN14, with identical isoelectric focusing patterns have the same biochemical properties. Chromosomal and R-factor-mediated beta-lactamases from single strains have been separated and their identities confirmed by immunoisoelectric focusing. R factor-mediated enzymes gave identical isoelectric focusing patterns irrespective of the host strain. Isoelectric focusing can therefore be used to observe the transfer of beta-lactamases carried by R factors.

Thirteen strains of the gram-negative, facultative phototrophic bacterium Rhodobacter sphaeroides were examined fro susceptibility to beta-lactam antibiotics. All strains were sensitive to the semisynthetic penicillins ampicillin, carbenicillin, oxacillin, cloxacillin, and methicillin, but 10 of the 13 strains were resistant to penicillin G, as well as a number of cephalosporins, such as cephalothin, cephapirin, and cephalosporin C. A beta-lactamase (EC 3.5.2.6) with strong cephalosporinase activity was detected in all of the resistant strains of R. sphaeroides. With strain Y-1 as a model, it was shown that the beta-lactamase was inducible by penicillin G, cephalosporin C, cephalothin, and to some minor extent, cephapirin. The beta-lactamase was located in the periplasmic space, from which it could be extracted by osmotic shock disruption. By using this fraction, the beta-lactamase was purified 34-fold to homogeneity by steps involving batch adsorption to and elution from DEAE-Sephadex A50, chromatography on Q-Sepharose, and preparative polyacrylamide gel electrophoresis. The molecular masses of the native and denatured enzymes were determined to be 38.5 kilodaltons by gel filtration and 40.5 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating a monomeric structure. The isoelectric point was estimated to be at pH 4.3. In Tris hydrochloride buffer, optimum enzyme activity was measured at pH 8.5. The beta-lactamase showed high activity in the presence of the substrates cephalothin, cephapirin, cephalosporin C, and penicillin G, for which the apparent Km values were 144, 100, 65, and 110 microM, respectively. Cephalexin, cepharidine, and cephaloridine were poor substrates. The beta-lactamase was strongly inhibited by cloxacillin and oxacillin but only slightly inhibited by phenylmethylsulfonyl fluoride or thiol reagents such as iodoacetate and p-chloromercuribenzoate.

The purified cephalosporinase from Proteus vulgaris hydrolyzed a variety of cephalosporins, including cefuroxime, at a high level; its activity was inhibited by clavulanic acid.

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.