SHV型β内酰胺酶新亚型SHV-71的发现

目的了解鲍曼不动杆菌中SHV型β内酰胺酶的基因型及其耐药性。方法采用纸片扩散法(K-B法)测定5株鲍曼不动杆菌对13种抗菌药物的敏感性;设计SHV型β内酰胺酶引物,采用PCR法获得SHV型酶编码基因的片段,并将PCR扩增产物克隆入pMD18-T载体,以双脱氧链终止法测定核苷酸序列,确定亚型。结果药敏试验结果显示:5株鲍曼不动杆菌对氨苄西林、哌拉西林、头孢西丁、头孢他啶和氨曲南均耐药。3株菌SHV基因型扩增结果呈阳性,其中2株与SHV-12的序列100%相同,另1株(No-03)经GenBank网上同源性比较,未发现完全相同的核苷酸和氨基酸序列,为一种新发现的SHV亚型β内酰胺酶耐药基因,其序列以SHV-71的名称在美国核酸数据库GenBank成功登录(GenBank注册号:DQ296194)。结论临床分离的鲍曼不动杆菌No-03株所产SHV型BLA为一种新SHV亚型β内酰胺酶。     

Survey and molecular genetics of SHV beta-lactamases in Enterobacteriaceae in Switzerland: two novel enzymes, SHV-11 and SHV-12.

Sixty isolates of Enterobacteriaceae resistant to beta-lactam antibiotics were collected over a period of 2 years in Switzerland and screened by hybridization for the carriage of SHV genes. Thirty-four positive strains were found, and their SHV genes were amplified and sequenced. SHV extended-spectrum beta-lactamases (ESBLs) were found: 13 strains contained SHV-2a, 12 harbored SHV-2, and SHV-5 was found twice. Four strains were shown to contain SHV-1. In addition, we report two new SHV variants, termed SHV-11 (non-ESBL) and SHV-12 (ESBL). In spite of the carriage of SHV ESBLs, many strains showed only low resistance to one or more third-generation cephalosporins. In addition, 26 did not transfer the blaSHV gene in mating experiments.     

Rapid discriminatory detection of genes coding for SHV beta-lactamases by ligase chain reaction

Ligase chain reaction (LCR) is a recently developed technique that employs a thermostable ligase and allows for the discrimination of DNA sequences differing in only a single base pair. The method has been adapted and applied to differentiation of bla(SHV) genes. We have developed an LCR typing method to characterize point mutations in genes for SHV-derived extended-spectrum beta-lactamases with four different sets of biotinylated LCR primers. To evaluate the applicability of the current technique, we tested seven Escherichia coli strains producing SHV-1, SHV-2, SHV-2a, SHV-3, SHV-4, SHV-5, and SHV-12. With the LCR typing, seven SHV genes can be distinguished according to their incorporating point mutations. In an attempt to characterize SHV beta-lactamases by LCR typing in clinical isolates, 46 strains carrying bla(SHV) genes (32 Klebsiella pneumoniae, 10 Enterobacter cloacae, and 4 E. coli) were subjected to antibiotic susceptibility testing, isoelectric focusing, and LCR typing. LCR typing allowed the characterization of beta-lactamases, and genotypes obtained by LCR typing were in accordance with phenotypes such as antibiotic resistance profile and pI value of beta-lactamase. Therefore, we concluded that LCR typing may permit defining the SHV families with simplicity and reliability and can be applied to the detailed characterization and molecular epidemiology of SHV-type beta-lactamases.     

Comparative activities of clavulanic acid, sulbactam, and tazobactam against clinically important beta-lactamases.

Clavulanic acid, sulbactam, and tazobactam are inhibitors of a variety of plasmid-mediated beta-lactamases. However, inhibition data for these three inhibitors with a wide range of different plasmid-mediated beta-lactamases have not yet been compared under the same experimental conditions. A number of groups have inferred that clavulanic acid inhibits extended-spectrum TEM and SHV beta-lactamases, but inhibition data have rarely been published. In this study, the 50% inhibitory concentrations of these three beta-lactamase inhibitors for 35 plasmid-mediated beta-lactamases have been determined. Of these 35 beta-lactamases, 20 were extended-spectrum TEM- or SHV-derived beta-lactamases. The other 15 enzymes were conventional-spectrum beta-lactamases such as TEM-1 and SHV-1. Clavulanic acid was a more potent inhibitor than sulbactam for 32 of the 35 plasmid-mediated beta-lactamases tested. In particular, clavulanic acid was 60 and 580 times more potent than sulbactam against TEM-1 and SHV-1, respectively, currently the two most clinically prevalent gram-negative plasmid-mediated beta-lactamases. Statistical analysis of the data of the 50% inhibitory concentrations showed that clavulanic acid was 20 times more active overall than sulbactam against the conventional-spectrum enzymes. In addition, clavulanic acid was 14 times more potent than sulbactam at inhibiting the extended-spectrum enzymes. Tazobactam also showed significantly greater activity than sulbactam against the two groups of beta-lactamases. There were no significant differences between the overall activities of tazobactam and clavulanic acid against the extended-spectrum TEM and SHV enzymes and conventional-spectrum enzymes, although differences in their inhibition profiles were observed.     

Amino acid residues that contribute to substrate specificity of class A beta-lactamase SME-1

Carbapenem antibiotics are used as antibiotics of last resort because they possess a broad spectrum of antimicrobial activity and are not easily hydrolyzed by beta-lactamases. Recently, class A enzymes, such as the SME-1, NMC-A, and IMI-1 beta-lactamases, have been identified with the capacity to hydrolyze carbapenem antibiotics. Traditional class A beta-lactamases, such as TEM-1 and SHV-1, are unable to hydrolyze carbapenem antibiotics and exhibit some differences in sequence from those that are able to hydrolyze carbapenem antibiotics. The positions that differ may contribute to the unique substrate specificity of the class A carbapenemase SME-1. Codons in the SME-1 gene representing residues 104, 105, 132, 167, 237, and 241 were randomized by site-directed mutagenesis, and functional mutants were selected for the ability to hydrolyze imipenem, ampicillin, or cefotaxime. Although several positions are important for hydrolysis of beta-lactam antibiotics, no single position was found to uniquely contribute to carbapenem hydrolysis. The results of this study support a model whereby the carbapenemase activity of SME-1 is due to a highly distributed set of interactions that subtly alter the structure of the active-site pocket.     

Extended-spectrum beta-lactamases in clinical isolates of Enterobacter gergoviae and Escherichia coli in China.

Resistance to ceftazidime, detected in isolates of Escherichia coli 5518 and Enterobacter gergoviae 3773 from our hospital, was transferred, together with resistance to aminoglycosides, trimethoprim, sulfonamide, and other beta-lactam antibiotics, by conjugation to E. coli JP559. Both E. coli transconjugants were resistant to ampicillin, all cephalosporins, and aztreonam but remained susceptible to cefoxitin and imipenem. The enzymes of the two transconjugant strains readily hydrolyzed cephalosporins in a spectrophotometric assay. Hybridization results suggested that the extended-spectrum beta-lactamase produced by E. coli 5518 was a non-TEM, non-SHV enzyme, the origin of which is currently unknown. The beta-lactamase produced by E. gergoviae 3773 was of the SHV type and was further proved to be SHV-2 by DNA sequencing. Thus, extended-spectrum beta-lactamases are occurring in China as well as in other parts of the world.     

Extended-spectrum beta-lactamases (ESBLs) producing bacteria

TEM- or SHV-type extended-spectrum beta-lactamases(ESBLs) are of clinical concern in Europe and the United States, whereas bacterial strains producing such types of ESBLs had not been reported in Japan for many years. Toho-1, a different type class A ESBL, has been reported in 1995, in which any prototypical enzyme has not been identified so far. At present Toho-1 is the major ESBL in Japan, however, SHV5 alpha has been reported in 1998, followed by TEM-26, SHV-2, and SHV12. More recently, SHV-24, a novel SHV-derived ESBL has also been found. Since Toho-1-type ESBL, AmpC-type beta-lactamase, and class B metallo-beta-lactamase have been widely found in Japan, a novel detection system for ESBLs suitable for this country should be developed.     

Diversity of SHV and TEM beta-lactamases in Klebsiella pneumoniae: gene evolution in Northern Taiwan and two novel beta-lactamases, SHV-25 and SHV-26

A total of 113 blood culture isolates of Klebsiella pneumoniae from 10 hospitals in northern Taiwan were studied for SHV and TEM beta-lactamase production. bla(SHV) was amplified from all isolates by PCR. TEM-type resistance, was found in 32 of the isolates and was of the TEM-1 type in all isolates. SHV-1, -2, -5, -11, and -12 and two novel enzymes were identified. These novel enzymes were designated SHV-25 and SHV-26 and had pIs of 7.5 and 7.6, respectively. Amino acid differences in comparison to the amino acid sequence of bla(SHV-1) were found at positions T18A (ThrACC-->AlaGCC), L35Q (LeuCTA-->GluCAA), and M129V (MetATG-->ValGTG) for SHV-25 and at position A187T (AlaGCC-->ThrACC) for SHV-26. The results of substrate profiles and MIC determinations showed that the novel enzymes did not hydrolyze extended-spectrum cephalosporins, rendering the isolates susceptible to these agents. Inhibition profiles revealed that the 50% inhibitory concentration for SHV-26 was higher than those for SHV-1 and SHV-25, resulting in an intermediate resistance to amoxicillin-clavulanic acid. Forty-nine ribotypes were identified, suggesting that major clonal spread had not occurred in any of the hospitals. According to the amino acid sequence, SHV beta-lactamases in Taiwan may basically be derived through stepwise mutation from SHV-1 or SHV-11 and further subdivided by four routes. The stepwise mutations initiated from SHV-1 or SHV-11 to SHV-2, SHV-5, and SHV-12 comprise the evolutionary change responsible for extended-spectrum beta-lactamase (ESBL) production in Taiwan. The stepwise mutations that lead to a non-ESBL (SHV-25) and the beta-lactamase (SHV-26) with reduced susceptibility to clavulanic acid are possibly derived from SHV-11 and SHV-1, respectively. The results suggest a stepwise evolution of SHV beta-lactamases in Taiwan.     

Amino acid substitutions causing inhibitor resistance in TEM beta-lactamases compromise the extended-spectrum phenotype in SHV extended-spectrum beta-lactamases

Three amino acid substitutions, Met-69-->Ile, Arg-244-->Ser and/or Asn-276-->Asp, mediate inhibitor resistance (IR) in TEM beta-lactamases. They were introduced in all possible combinations at homologous positions into either SHV-1 or the respective extended-spectrum beta-lactamases (ESBLs), SHV-2 or SHV-5. Susceptibility testing of the resulting set of seven variants of each parental strain, all in an isogenic background, was performed. The phenotypes of the constructions revealed that most substitutions resulted in reduced resistance to most tested single beta-lactam formulations. This decrease over-compensated for the expected increase in inhibitor resistance, so that most mutants showed no rise in resistance to inhibitor/beta-lactam combinations, although increases of MICs from one- to 43-fold compared with the respective parental strains were also measured. Combination of several IR-determining substitutions impaired both phenotypes in the carrier strains even more. None of the 14 mutants derived from the ESBLs, SHV-2 and SHV-5, showed a clinically relevant combined ESBL-IR phenotype. These findings indicate that the SHV beta-lactamase does not benefit proportionally from simultaneous substitution of residues relevant for the ESBL and the IR phenotype.     

Molecular analysis of the simultaneous production of two SHV-type extended-spectrum beta-lactamases in a clinical isolate of Enterobacter cloacae by using single-nucleotide polymorphism genotyping

Bacteria that simultaneously produce multiple extended-spectrum beta-lactamases are frequently isolated. We report an Enterobacter cloacae isolate, ES24, producing four different beta-lactamases (AmpC type beta-lactamase, TEM-1, SHV-7, and a novel extended-spectrum beta-lactamase, SHV-30). Direct sequencing of bla(SHV) gene products gave a "double peak" at position 703, suggesting the presence of more than one allele. Using fluorescence resonance energy transfer real-time PCR to detect single-nucleotide polymorphisms, we were able to distinguish two different bla(SHV) genes in a single isolate. This may prove to be a useful technique in surveys of beta-lactamase production in contemporary clinical isolates.     

Discrimination of SHV β-Lactamase Genes by Restriction Site Insertion-PCR

Restriction site insertion-PCR (RSI-PCR) is a simple, rapid technique for detection of point mutations. This technique exploits primers with one to three base mismatches near the 3' end to modulate a restriction site. We have developed this technique to identify described mutations of the bla(SHV) genes for differentiation of SHV variants that cannot be distinguished easily by other techniques. To validate this method, eight standard strains were used, each producing a different SHV beta-lactamase: SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, SHV-6, SHV-8, and SHV-18. Mismatch primers were designed to detect mutations affecting amino acids at positions 8 (SspI), 179 (HinfI), 205 (PstI), 238 (Gly-->Ala) (BsrI), and 240 (NruI) of bla(SHV) genes. All amplimers of the bla(SHV) genes used in this study yielded the predicted restriction endonuclease digestion products. In addition, this study also makes theoretical identification of bla(SHV-6), bla(SHV-8), and 12 novel bla(SHV) variants using the PCR-restriction fragment length polymorphism (RFLP) technique possible. By using a combination of PCR-RFLP and RSI-PCR techniques, up to 27 SHV variants can now be distinguished rapidly and reliably. These simple techniques are readily applied to epidemiological studies of the SHV beta-lactamases and may be extended to the characterisation of other resistance determinants.     

The emergence of beta-lactamase resistance in respiratory pathogens

Bacteria have evolved a variety of mechanisms to express resistance to beta-lactam antibiotics. beta-Lactamase-induced hydrolysis of the beta-lactam ring is the principal and most important mediator of clinically significant resistance. Almost 200 beta-lactamases have now been identified, of which class 1 chromosomal beta-lactamases, class 2b plasmid-mediated beta-lactamases (especially TEM-1) and class 2be extended-spectrum beta-lactamases (ESBLs) are among the most important in respiratory pathogens. The combination of enzymatic and non-enzymatic resistance mechanisms has led to a steady rise in the prevalence of resistance to beta-lactams among isolates of the major respiratory pathogens, and, in turn, increasing rates of treatment failure, increased mortality, and prolonged morbidity. The combination of a beta-lactam antibiotic with a beta-lactamase inhibitor such as sulbactam, which protects the antibiotic from beta-lactamase destruction and so restores its activity, provides an innovative solution to this problem.     

SHV-5, a novel SHV-type beta-lactamase that hydrolyzes broad-spectrum cephalosporins and monobactams.

SHV-5 (pI 8.2), a novel broad-spectrum beta-lactamase encoded by a ca. 150-kilobase plasmid, was found in Klebsiella pneumoniae 160. SHV-5 beta-lactamase caused decreased susceptibility to most penicillins, cephalosporins, and monobactams, except imipenem and compounds which have a C6 or C7 alpha-methoxy substituent. beta-Lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) inhibited its activity and showed a synergistic effect when associated with different hydrolyzable beta-lactam compounds. Hybridization studies suggested that this enzyme may be related to, or derived from, the SHV enzyme. Increased MICs of cephamycins and temocillin associated with a decreased synergistic effect of the inhibitors on K. pneumoniae 160 might be linked to a decrease in two outer membrane proteins.     

Characterization of the plasmid mediated beta-lactamase BIL-1.

A multi-resistant strain of Escherichia coli isolated from a patient from Pakistan was shown to possess a new plasmid-mediated beta-lactamase. This enzyme, designated BIL-1, conferred resistance to extended-spectrum cephalosporins such as cefotaxime and ceftazidime. However, unlike other plasmid-mediated beta-lactamases capable of conferring resistance to these drugs, the BIL-1 was not a member of the TEM or SHV group of plasmid-mediated beta-lactamases and it also conferred resistance to beta-lactam drugs in combination with beta-lactamase inhibitors (i.e. clavulanic acid). The biochemical properties of the enzyme suggest that BIL-1 is related to the Richmond & Sykes Class I chromosomal beta-lactamases. Its inhibition properties by various beta-lactam drugs are similar to the inhibition properties of the chromosomally-encoded P99 enzyme of Enterobacter cloacae.     

Comparative potency of mecillinam and other beta-lactam antibiotics against Escherichia coli strains producing different beta-lactamases

The activity of mecillinam, a beta-lactam antibiotic with high affinity for gram-negative penicillin-binding protein 2 (PBP2), was assessed against ampicillin-resistant Escherichia coli strains producing beta-lactamases representing the three molecular classes, A (TEM-1 and -3, SHV-3 and IRT-5), C (AmpC) and D (OXA-3). The antimicrobial activity of mecillinam and other beta-lactam antibiotics was evaluated by determining their MICs on Mueller-Hinton agar. The time course of hydrolysis in crude extracts prepared from the various beta-lactamase-producing strains was also measured and was used to determine the relative rate of hydrolysis and the apparent affinity for ampicillin, cephalothin and mecillinam. When compared with the other beta-lactam antibiotics, mecillinam demonstrated significantly greater antibacterial potency and higher stability to beta-lactamase hydrolysis in TEM-, IRT- and AmpC-producing isolates. These findings confirm that the antimicrobial potency of mecillinam compares favourably with those of the other penicillins included in the present study, suggesting that mecillinam use in the treatment of infections caused by gram-negative bacteria should be re-evaluated.     

Use of an isogenic Escherichia coli panel to design tests for discrimination of beta-lactamase functional groups of Enterobacteriaceae

A study was designed to determine if an isogenic panel of Escherichia coli strains containing many different beta-lactamases could be used for the preliminary screening of a large number of beta-lactam agents to identify which might be most useful in the development of a definitive test for specific beta-lactamases found among the members of family Enterobacteriaceae. The susceptibilities of 46 strains, comprising the isogenic panel, to expanded-spectrum cephalosporins, cephamycins, and aztreonam were determined in the presence and absence of beta-lactamase inhibitors in broth microdilution tests. The results indicated that strains producing extended-spectrum beta-lactamases (ESBLs) could be distinguished from strains producing other Bush-Jacoby-Medeiros functional group 2 or group 1 beta-lactamases. For strains producing group 1 beta-lactamases, cefpodoxime and ceftazidime MICs were > or = 4 micrograms/ml and addition of clavulanate did not reduce the MICs more than fourfold. For strains producing group 2 enzymes other than ESBLs, cefpodoxime and ceftazidime MICs were < or = 2 micrograms/ml. With a single exception (ceftazidime for the strain producing SHV-3), among strains producing ESBLs, cefpodoxime and ceftazidime MICs were > or = 4 micrograms/ml and addition of clavulanate reduced the MICs by more than eightfold. Cephamycins could also be used to discriminate between strains producing group 1 beta-lactamases and ESBLs, since only the former required cefotetan concentrations as high as 8 micrograms/ml or cefoxitin concentrations of > 16 micrograms/ml for inhibition. Other cephalosporins provided some discrimination between the various beta-lactamase producers, although they were not as reliable as either cefpodoxime or ceftazidime. These results indicate the utility of an isogenic panel for identification of candidate drugs among many for further testing with clinical isolates of the family Enterobacteriaceae to determine the best agents for detection of specific beta-lactamases in this family.     

In-vitro selection of porin-deficient mutants of two strains of Klebsiella pneumoniae with reduced susceptibilities to meropenem, but not to imipenem

We have evaluated the ability of imipenem and meropenem to select, in vitro, resistant mutants of two clinical isolates of Klebsiella pneumoniae producing both SHV and TEM beta-lactamases. Only meropenem selected mutants of both isolates for which the MICs of meropenem, but not imipenem, were markedly higher than those for the parent strains; the MICs of several other beta-lactam antibiotics, including beta-lactam/beta-lactamase inhibitor combinations, for these mutants were also higher than those for the parent strains. In contrast, the MICs for the imipenem-selected mutants were the same as, or similar to, those for the parent strains. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed that an outer membrane protein in both parent strains was absent in the meropenem-selected mutants, but not in the imipenem-selected mutants. This protein is likely to be a porin, the absence of which is presumably associated with impaired beta-lactam permeability and, therefore, the reduced susceptibilities to these antibiotics exhibited by the mutant strains. We believe that this is the first report of the in-vitro selection of porin-deficient mutants of K. pneumoniae following exposure to meropenem.     

Combating bacterial resistance in skin and skin-structure infection: importance of beta-lactamase inhibition.

Serious skin and skin-structure infections may require parenteral antibiotic therapy. Such infections are generally polymicrobial, and they often involve both gram-positive and gram-negative aerobes as well as anaerobic bacteria. Effective treatment thus requires the use of a broad-spectrum antibiotic or combination therapy. The development of antibiotic resistance by clinically important pathogens has significantly increased the difficulty of treating skin and skin-structure infections. One of the major mechanisms of resistance observed in organisms likely to be associated with such infections is the development of beta-lactamases that inactivate beta-lactam antibiotics. Two approaches have been taken to combat this problem: the use of beta-lactam/beta-lactamase inhibitor combinations and the development of beta-lactamase-stable drugs. Both strategies have resulted in treatments that are clinically and bacteriologically effective in patients with skin and skin-structure infections. The use of one beta-lactam/beta-lactamase inhibitor combination, ampicillin/sulbactam, has been demonstrated to be more cost-effective than treatment with beta-lactamase-stable antibiotics, such as cefoxitin and imipenem/cilastatin, for this indication.     

Use of Microdilution Panels with and without β-Lactamase Inhibitors as a Phenotypic Test for β-Lactamase Production among Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter freundii, and Serratia marcescens

Over the past decade, a number of new beta-lactamases have appeared in clinical isolates of Enterobacteriaceae that, unlike their predecessors, do not confer beta-lactam resistance that is readily detected in routine antibiotic susceptibility tests. Because optimal methodologies are needed to detect these important new beta-lactamases, a study was designed to evaluate the ability of a panel of various beta-lactam antibiotics tested alone and in combination with beta-lactamase inhibitors to discriminate between the production of extended-spectrum beta-lactamases, AmpC beta-lactamases, high levels of K1 beta-lactamase, and other beta-lactamases in 141 isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, and Serratia marcescens possessing well-characterized beta-lactamases. The microdilution panels studied contained aztreonam, cefpodoxime, ceftazidime, cefotaxime, and ceftriaxone, with and without 1, 2, and 4 microg of clavulanate per ml or 8 microg of sulbactam per ml and cefoxitin and cefotetan with and without 8 microg of sulbactam per ml. The results indicated that a minimum panel of five tests would provide maximum separation of extended-spectrum beta-lactamase high AmpC, high K1, and other beta-lactamase production in Enterobacteriaceae. These included cefpodoxime, cefpodoxime plus 4 microg of clavulanate per ml, ceftazidime, ceftriaxone, and ceftriaxone plus 8 microg of sulbactam per ml. Ceftriaxone plus 2 microg of clavulanate per ml could be substituted for cefpodoxime plus 4 microg of clavulanate per ml without altering the accuracy of the tests. This study indicated that tests with key beta-lactam drugs, alone and in combination with beta-lactamase inhibitors, could provide a convenient approach to the detection of a variety of beta-lactamases in members of the family Enterobacteriaceae.     

Influence of clindamycin on derepression of beta-lactamases in Enterobacter spp. and Pseudomonas aeruginosa.

Previous studies in this and other laboratories have shown that derepression of beta-lactamases in strains of Enterobacter and Pseudomonas spp. is responsible for the rapid development of resistance to a variety of beta-lactam antibiotics. The purpose of the current study was to evaluate the effects of clindamycin on derepression of beta-lactamases in these two genera. In tests with four strains of each genus, clindamycin diminished derepression in one isolate of each genus and completely prevented derepression in a second Enterobacter isolate (strain 55). Additional tests with strain 55 revealed that other inhibitors of macromolecular synthesis did not completely prevent derepression of beta-lactamase when tested at concentrations that did not inhibit replication. However, clindamycin did not affect synthesis of beta-lactamase that was constitutively produced in a mutant of this strain (55M). It also did not inhibit derepression of beta-galactosidase in either strain 55 or 55M. Clindamycin did not diminish the bactericidal effects of beta-lactam antibiotics against Enterobacter or Pseudomonas spp. However, it enhanced the bactericidal activity of cefamandole against strain 55. These in vitro effects of clindamycin on strain 55 that were related to prevention of derepression of beta-lactamase were confirmed in vivo with an animal model of infection. These results indicate that in some strains, clindamycin can specifically prevent derepression of beta-lactamases without inhibiting growth. Such a selective effect may provide a new approach for the enhancement of the antibacterial activity of certain beta-lactam antibiotics.