Plasmid-mediated Enzymes Research Articles

Resistance to and Immunomodulation Effects of Cephalosporin Antibiotics

In order to be effective, cephalosporins must penetrate the outer membrane of bacteria to reach the β-lactam target enzymes, avoid hydrolysis by β-lactamases and bind to the bacterial penicillin-binding proteins (PBPs) corresponding to the peptidases responsible for catalysing the cross-linking of peptidoglycan. Resistance to cephalosporins can arise if the targets are modified, or if they are protected by other cell products (β-lactamases) or structures (permeability barriers). Target modification can involve either reduced affinity of an existing PBP or the acquisition of supplementary β-lactam-resistant PBPs. β-Lactamases are produced universally by bacteria and may be chromosomally or plasmid-mediated. Although generally species specific, chromosomal enzymes from Gram-negative bacteria can be divided into 4 main categories: chromosomal cephalosporinases (not inhibited by clavulanic acid), cefuroximases, broad spectrum β-lactamases, and β-lactamases of anaerobic species. Plasmid-mediated enzymes include 3 categories: penicillinases from Staphylococcus aureus, broad spectrum β-lactamases (e.g. TEM-1, -2; SHV-1) and extended broad spectrum β-lactamases (TEM-3, -5; SHV-2 to SHV-5). The degree of β-lactamase-mediated resistance is related to the amount of enzyme produced, the location of the enzyme and the kinetics of the enzyme’s activity. β-Lactamases of Gram-positive organisms are usually extracellular, while those of Gram-negative bacteria are retained within the cell periplasm. While synthesis of β-lactamases by streptococci is of little or no clinical importance, these enzymes play a major role in the resistance of staphylococci. The outer membrane of Gram-negative organisms constitutes a substantial permeability barrier retarding the entry of β-lactams into the cell. Nevertheless, specialised outer membrane proteins called ‘porins’ create pores in this membrane, enabling the diffusion of small aqueous solutes, such as cephalosporins. Shielding by permeability barriers is minimal in Gram-positive bacteria, as the PBPs of such organisms are located on the outer aspect of the cytoplasmic membrane.

Chemical modification of tazobactam. Synthesis of 2 beta-[(4-substituted)-1,2,3-triazol-1-yl]methyl penicillanic acid sulfone derivatives.

A series of 2 beta-[(4-substituted)-1,2,3-triazol-1-yl] methyl penicillanic acid sulfones was synthesized as beta-lactamase inhibitors. Many of these compounds showed good in vitro inhibitory activity against penicillinase, cefotaximase and plasmid-mediated class III TEM enzymes, but exhibited weaker cephalosporinase inhibition. One member in this series--2 beta-[(4-pyridiniummethyl)-1,2,3-triazol- 1-yl]-6,6-dihydropenicillanate 1,1-dioxide (12a), when tested in combination with piperacillin, showed excellent synergistic activity against microorganisms producing plasmid-mediated enzymes, but had insufficient activity against microorganisms producing chromosomally mediated class I enzymes.

Beta-Lactamase hydrolysis of cephalosporin 3'-quinolone esters, carbamates, and tertiary amines.

The beta-lactam hydrolysis of five cephalosporin 3'-quinolones (dual-action cephalosporins) by three gram-negative beta-lactamases was examined. The dual-action cephalosporins tested were the ester Ro 23-9424; the carbamates Ro 25-2016, Ro 25-4095, and Ro 25-4835; and the tertiary amine Ro 25-0534. Also tested were cephalosporins with similar side chains (cefotaxime, desacetylcefotaxime, cephalothin, cephacetrile, and Ro 09-1227 [SR 0124]) and standard beta-lactams (penicillin G, cephaloridine). The beta-lactamases used were the plasmid-mediated TEM-1 and TEM-3 enzymes and the chromosomal AmpC. The cephacetrile-related compounds Ro 25-4095 and Ro 25-4835 were hydrolyzed by all three beta-lactamases with catalytic efficiencies (relative to penicillin G) ranging from approximately 5 (TEM-1, AmpC) to approximately 25 (TEM-3). The cephalothin-related Ro 25-2016 was also hydrolyzed by all three beta-lactamases, particularly the AmpC enzyme (relative catalytic efficiency, 110). The cefotaxime-related compounds Ro 25-0534 and Ro 23-9424 were hydrolyzed to any significant extent only by the TEM-3 enzyme (relative catalytic efficiencies, 1.2 and 4.7, respectively.

Phylogenetic Tree and Sequence Similarity of β-Lactamases

β-Lactamases are the main cause of β-lactam resistance in many pathogenic bacteria. These enzymes can be detected in a variety of pathogenic as well as non-pathogenic bacteria. The cyanobacteria are also known to produce a β-lactamase. Recently, the amino acid sequences and the three-dimensional structures of some of these β-lactamases have been clarified. On the basis of the amino acid sequences of 47 β-lactamases and the computer-aided analysis, a phylogenetic tree is proposed in this paper. According to the tree, β-lactamases are classified into six groups. Group 1 β-lactamases are mainly composed of plasmid-mediated enzymes from gram-negative bacteria. However, chromosome-derived β-lactamases from Klebsiella pneumoniae and Rhodopseudomonas capsulata take part in this group. Group 2 enzymes consist of a part of the chromosome-encoded β-lactamases from Streptomyces, and chromosome-mediated enzymes from Yersinia enterocolitica, Citrobacter diversus, and Klebsiella oxytoca. Chromosome-encoded β-lactamases from gram-negative bacteria form group 3. Group 4 is composed of metalloenzymes, whereas group 5 consists of OXA type β-lactamases. Chromosome-encoded β-lactamases from gram-positive bacteria form group 6. Comparison of the amino acid sequences among these groups confirmed the phylogenetic tree and the classification: the β-lactamases in each group have its particular conserved amino acid sequences. In addition, the tree provides more detailed classification and time-scale mutual relationships and predicts new types of β-lactamases that may be found. Furthermore, the classification deduced from the tree is generally in accord with the one based on the amino acid sequences reported previously. However, the class A β-lactamases are clearly divided into three groups: groups 1, 2, and 6. RDF2 analysis shows that some combinations between β-lactamases and β-lactam-interacting proteins as well as eukaryotic proteins have a low but significant evolutionary relatedness.

Importance of beta-lactamase stability in treating today's respiratory tract infections.

In respiratory tract infections Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae and Klebsiella spp. are the most frequently encountered bacterial pathogens. Resistance of clinical S. pneumoniae isolates is known to be independent of beta-lactamase production, whereas resistance of the other species mentioned is due to beta-lactamase production. With respect to M. catarrhalis the first beta-lactamase-producing (bla+) isolate was detected in clinical specimens in 1976 and now, 70-90% of all clinical isolates are bla+. The enzymes BRO-1 and BRO-2 are transposon-mediated thus explaining their rapid spread; they hydrolyze penicillin compounds very rapidly and, to a lesser extent, the older cephalosporins. Resistance in clinical H. influenzae isolates is mainly due to the prevalence of the most widespread transposon-mediated beta-lactamase, TEM-1, which has a substrate profile resembling that of the BRO enzymes. Bla+ H. influenzae isolates make up to 6% of the total in Northern Europe, in Southern Europe up to 55% and in many African countries more than 80%. More than 95% of Klebsiella spp. isolates possess a chromosomally encoded penicillinase and to a varying extent a plasmid-mediated enzyme in addition. Recently, reports from several countries have pointed to the clinical relevance of 'extended-spectrum enzymes' derived by point mutation from the 'classical' TEM-1 or TEM-2 enzymes. These new enzymes (TEM-3 to TEM-21) exhibit a broadened substrate profile, inactivating even the oxyiminocephalosporins. The most stable compounds are ceftibuten and cefetamet. With respect to the future, these enzymes may spread between species due to their location on transposons.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of chromosomally encoded penicillinases in clinical isolates of Klebsiella pneumoniae.

Four Klebsiella pneumoniae strains, isolated in two geographically distant French hospitals, were found to produce constitutive beta-lactamases with an unusual isoelectric point for this species (8.1). The four enzymes were chromosomally encoded and related to the Ambler's class A plasmid-mediated SHV-type enzymes. The four enzymes were predominantly penicillinases, with good activity against amino- and ureidopenicillins. They poorly hydrolysed cephalothin, cephaloridine and cefoperazone and did not hydrolyse third-generation cephalosporins and aztreonam. The four enzymes were susceptible to the inhibitory effect of clavulanic acid. Finally, the strains were all found to produce an acetyl-esterase. The acetyl-esterases catalysized hydrolysis of the acetoxy group of cephalothin and cefotaxime although this did not affect their antibacterial activities. These esterases were not susceptible to the inhibitory effect of clavulanic acid and EDTA.

Effect of clavulanic acid on activity of beta-lactam antibiotics in Serratia marcescens isolates producing both a TEM beta-lactamase and a chromosomal cephalosporinase

An isolate of Serratia marcescens that produced both an inducible chromosomal and a plasmid-mediated TEM-1 beta-lactamase was resistant to ampicillin and amoxicillin and also demonstrated decreased susceptibility to extended-spectrum beta-lactam antibiotics (ESBAs). Clavulanic acid did not lower the MICs of the ESBAs, but it decreased the MICs of the penicillins. The TEM-1-producing plasmid was transferred to a more susceptible S. marcescens strain that produced a well-characterized inducible chromosomal beta-lactamase. The MICs of the ESBAs remained at a low level for the transconjugant. Ampicillin and amoxicillin which were good substrates for the plasmid-mediated enzyme, were not well hydrolyzed by the chromosomal enzymes; the ESBAs were hydrolyzed slowly by all the enzymes. When each of the S. marcescens strains was grown with these beta-lactam antibiotics, at least modest increases in chromosomal beta-lactamase activity were observed. When organisms were grown in the presence of clavulanic acid and an ESBA, no enhanced induction was observed. The increases in the MICs of the ESBAs observed for the initial clinical isolate may have been due to a combination of low inducibility, slow hydrolysis, and differences in permeability between the S. marcescens isolates. When clavulanic acid and a penicillin were added to strains that produced both a plasmid-mediated TEM and a chromosomal beta-lactamase, much higher levels of chromosomal beta-lactamase activity were present than were observed in cultures induced by the penicillin alone. This was due to the higher levels of penicillin that were available for induction as a result of inhibition of the TEM enzyme by clavulanate.

Cefpodoxime: Comparable Evaluation with Other Orally Available Cephalosporins With a Note on the Role of β-Lactamases

The antibacterial activity of cefpodoxime, a new orally active methoxy-imino cephalosporin was evaluated in 470 recent isolates of gram-positive cocci and gram-negative rods from clinical specimens and compared to that of other orally active beta-lactam compounds. Cefpodoxime was highly active against ampicillin-resistant enterobacteria producing the plasmid-mediated TEM-1, TEM-2 or OXA-1 enzymes, as was the case for the other newer compounds. However, it was poorly active against cefuroxime-resistant (MIC greater than or equal to 16 mg/l) E. coli isolates, thus resembling cefetamet and cefixime. It was inactive against isolates exhibiting a production of large amounts of class I beta-lactamase, as was the case with all other compounds studied. Cefpodoxime was highly active against beta-hemolytic streptococci and against Haemophilus influenzae, resembling the related agents. Moreover, its activity against Staphylococcus aureus was comparable to that of cefotaxime and exceeded that of cefetamet and cefixime. Cefpodoxime and the other methoxyimino cephalosporins exhibited a poor affinity to the plasmid-mediated TEM-2 and OXA-1 enzymes. The hydrolysis of cefpodoxime by class I beta-lactamases was barely detectable, whereas it served as a moderate substrate for the enzyme from Klebsiella oxytoca 3951. Cefpodoxime, cefetamet and cefixime were slowly inactivated by the enzyme from Proteus vulgaris 4917 (an enzyme with cefuroximase activity) and much poorer substrates than cefotaxime.

Studies to optimize the in vitro testing of piperacillin combined with tazobactam (YTR 830)

The combination of piperacillin and the beta-lactamase inhibitor tazobactam (formerly YTR 830) was studied to determine optimal disk concentrations and dilution testing conditions. In addition, the potency of the combination was compared to that of piperacillin alone. The spectrum of piperacillin was greatly expanded by the addition to tazobactam principally against beta-lactamase producing strains of Haemophilus influenzae, Escherichia coli, Morganella morganii, Proteus vulgaris, Providencia stuartii, Shigella spp., Neisseria gonorrhoeae, and Staphylococcus spp. Tazobactam was active alone against Branhamella catarrhalis (minimum inhibitory concentration [MIC] 50, ⩽1 μg/ml), gonococci (MIC 50, 0.5–4 μg/ml), and N. meningitidis (MIC 50, ⩽1 μg/ml). Studies with beta-lactamase-producing type strains showed tazobactam to have high affinity for plasmid-mediated enzymes (TEM-1 and 2, SHV-1, HMS-1, and some CARB or OXA types) and not chromosomal beta-lactamases. Piperacillin/tazobactam inhibited 93% of fluoro-quinolone resistant strains at ⩽ 64 8 μ g/ml but failed to suppress the growth of 15 strains producing stably derepressed cephalosporinases. Comparisons of piperacillin/tazobactam results determined with 100 10 -, 100 20 -, and 100 30 -μ g disks established the 100 10 -μ g disk as most usable. Among five different MIC combinations the ratio of eight parts piperacillin to one part tazobactam or fixed concentration tests at ⩾4 μg tazobactam/ml were preferred, each producing very low occurrences (≤1.6%) of false-resistance or -susceptibility when compared to disk test results. MICs determined by agar and broth microdilution methods were essentially the same. The recommended breakpoints for piperacillin/tazobactam MICs were identical to those now found in the NCCLS susceptibility testing standards with the following exceptions: (1) for tests with H. influenzae and Staphylococcus spp.—susceptible at ⩾21 mm ( MIC ⩽ 16 2 μ g/ml ) and resistant ⩽20 mm ( MIC ⩾ 32 4 μ g/ml ); and (2) all remaining nonpseudomonas isolates would be interpreted by the NCCLS piperacillin enteric bacilli susceptibility criteria. This newer beta-lactamase inhibitor combination appears to be worthy of further in vivo trials guided by these or similar tentative in vitro susceptibility testing parameters.

Comparative in vitro and in vivo activities of piperacillin combined with the beta-lactamase inhibitors tazobactam, clavulanic acid, and sulbactam

Tazobactam (YTR-830H), a novel beta-lactamase inhibitor, was compared with clavulanic acid and sulbactam for enhancement of the activity of piperacillin against beta-lactamase-producing, piperacillin-resistant clinical isolates. Piperacillin MICs were determined in media containing a fixed concentration of 2 or 4 micrograms of the inhibitors per ml. The higher concentration was generally more effective. Tazobactam was superior to sulbactam in enhancing the spectrum and potency of piperacillin. Although the calvulanic acid combination was more potent, tazobactam was effective for a similar spectrum of resistant gram-negative clinical isolates containing beta-lactamase. MICs were reduced to the susceptible range for Escherichia coli, Klebsiella pneumoniae, Proteus spp., Salmonella spp., and Shigella spp. Combinations with tazobactam and sulbactam, but not clavulanic acid, were effective against Morganella spp. Some antagonism of the activity of piperacillin was observed with clavulanic acid but not with tazobactam or sulbactam. The inhibitors were similarly effective with piperacillin against beta-lactamase-positive Staphylococcus spp. and the Bacteroides fragilis group. Piperacillin-tazobactam was more effective against a broader spectrum of gram-negative enteric bacteria than ticarcillin plus clavulanic acid was. Combinations with tazobactam or clavulanic acid had a broader spectrum of activity than combinations with sulbactam against bacteria that produce characterized plasmid-mediated enzymes of clinical significance. In particular, piperacillin with tazobactam or clavulanic acid, but not with sulbactam, inhibited TEM-1, TEM-2, and SHV-1 enzymes. In vitro activity was reflected in vivo. Tazobactam and clavulanic acid were superior to sulbactam in enhancing the therapeutic efficacy of piperacillin in mice infected with beta-lactamase-positive E. coli, K. pneumoniae, Proteus mirabilis, and Staphylococcus aureus. Only combinations with tazobactam and sulbactam were effective against the Morganella infection. Tazobactam has a good potential for enhancing the clinical efficacy of piperacillin.

The β-Lactamases of Branhamella catarrhalis

Branhamella catarrhalis (B cat) has recently emerged as a common pathogen in the sputa of patients with chronic lung disease and has become an accepted cause of pneumonia. Drug therapy for B cat is influenced by the frequent production of β-lactamase (βla) by clinical strains. Two closely related βla (formerly Ravasio and 1908 and now called BRO-1 and BRO-2) are found in B cat and appear unique from known plasmid-mediated enzymes. We screened strains of B cat recovered in the United States since 1952 and identified no enzyme-positive isolates prior to August of 1976. The prevalence of the enzymes has risen dramatically since then, as βla production in 400 clinical isolates from our hospital has averaged 75% since βla studies were begun in 1983. BRO-1 and BRO-2 were also detected in two human upper respiratory species of the closely related subgenus Moraxella, M lacunata, and M nonliquefaciens, with the earliest isolate recovered in 1978. Using a filter mating technique, βla from B cat, M nonliquefaciens, and M lacunata was successfully transferred to two B cat recipients. Plasmid bands were demonstrated on agarose gel electrophoresis in 9/10 M nonliquefaciens and 2/9 B cat. However, no common plasmid was seen in βla-positive strains and no change in plasmid profiles was seen in βlapositive transconjugants. This supports previous studies suggesting that the βla genes are chromosomal. The BRO enzymes are a new finding in United States isolates of B cat, and appear to have been introduced into closely related Moraxella sp. at approximately the same time. They are unique from all known plasmid-mediated enzymes and are transferable, but do not appear to be plasmid mediated. Their genetic relatedness to other βla and their microbiologic source remain unknown.

Plasmid-mediated beta-lactamases among aminoglycoside resistant gram-negative bacilli.

Plasmid-mediated beta-lactamases were characterized by DNA hybridization in 371 aminoglycoside resistant gram-negative bacilli with known aminoglycoside resistance mechanism. Positive hybridization was detected in 50% to a TEM-1 probe, in 2% to a SHV-1 probe, and in 3% to both probes simultaneously. No hybridization was obtained to OXA-1, OXA-2, PSE-1/PSE-4/CARB-3 or PSE-2 beta-lactamase probes. TEM-1 beta-lactamase occurred simultaneously in 82% of strains showing the AAC(3)-V type of aminoglycoside resistance mechanism. Using isoelectric focusing as a control method, we found potentially plasmid-encoded beta-lactamases, other than TEM-1 and SHV-1, at various pIs in 13% of 288 randomly selected strains. The pIs of these strains or strains showing positive hybridizations did not fit to pIs of recently characterized plasmid-mediated enzymes against third-generation cephalosporins (e.g. CTX-1). In addition, the strains did not show resistance to cefotaxime or ceftazidime. According to the in vitro susceptibility data ceftazidime and cefotaxime were active against most of the aminoglycoside resistant strains studied. In contrast, the activity of piperacillin was much lower than that of the cephalosporins tested.

Interactions of meropenem, with beta-lactamases, including enzymes with extended-spectrum activity against third-generation cephalosporins.

The interactions of a meropenem were studied with a set of beta-lactamases including the new TEM- and SHV-related plasmid-mediated enzymes that have extended-spectrum activity against third-generation cephalosporins ('cefotaximases' and 'ceftazidimases'). Meropenem and imipenem were highly resistant to the hydrolytic activity of all the TEM and SHV related beta-lactamases, and to the OXA enzymes, as were the cephamycins: cefoxitin and cefotetan. The two carbapenems were also highly stable to Class C beta-lactamases (chromosomal cephalosporinases) whereas third-generation cephalosporins and cephamycins were slowly hydrolyzed. Both carbapenems demonstrated quite similar affinities for all the enzymes studied. In some instances, and particularly with Class A (TEM- and SHV-derived) enzymes, meropenem inactivated the beta-lactamase activity. Imipenem appeared less reactive in this respect.

Kinetic properties of two plasmid-mediated beta-lactamases from Klebsiella pneumoniae with strong activity against third-generation cephalosporins.

We determined the kinetic constants for two plasmid-mediated beta-lactamases with strong activity against third-generation cephalosporins: CTX-1 and SHV-2. The enzymes had many similar properties: their synthesis was constitutive and they were significantly active against penicillins as well as cephalosporins. The two enzymes thus differed considerably from the chromosomal cephalosporinases, but bore some resemblance to the commonly-encountered plasmid-coded penicillinases, such as TEM beta-lactamases. Moreover, like the TEM enzymes, the plasmid-mediated CTX-1 and SHV-2 enzymes were highly sensitive to the action of the inhibitors clavulanic acid and sulbactam. These inhibitors protected cefotaxime from hydrolysis by these enzymes. Both CTX-1 and SHV-2 lacked activity against the cephamycins, cefoxitin, latamoxef (moxalactam) and cefotetan. The CTX-1 and SHV-2 enzymes had a low activity against oxacillin and were not sensitive to chloride ions. Thus, they were not related to the OXA type beta-lactamases. For the third-generation cephalosporins the rates of hydrolysis were high and thus bore no relation with those observed for the other presently-known beta-lactamases, with perhaps the exceptions of those produced by K. oxytoca. Imipenem was very resistant to the action of these CTX-1 and SHV-2 beta-lactamases.

Mechanisms of Resistance to Cephalosporin Antibiotics

Cephalosporins, like other beta-lactams, bind to the bacterial penicillin-binding proteins (PBPs). These correspond to the D-ala-D-ala trans-, carboxy- and endo-peptidases responsible for catalysing the cross-linking of newly formed peptidoglycan. Resistance arises when the PBPs-and particularly the transpeptidases-are modified, or when they are protected by beta-lactamases or 'permeability barriers'. Target-mediated cephalosporin resistance can involve either reduced affinity of an existing PBP component, or the acquisition of a supplementary beta-lactam-insensitive PBP. beta-lactamases are produced widely by bacteria and may be determined by chromosomal or plasmid DNA. The chromosomal beta-lactamases are species-specific, but can be classified into a few broad groups. The plasmid-mediated enzymes cross interspecific and intergeneric boundaries. The level of beta-lactamase-mediated resistance relates to the amount of enzyme produced with or without induction; to the location of the enzyme (extracellular for Gram-positive organisms and periplasmic in Gram-negative ones); and to the kinetics of the enzyme's activity. In Gram-positive organisms the PBPs are located on the outer aspect of the cytoplasmic membrane and so shielding by permeability barriers is minimal. In Gram-negative cells, however, the PBPs are protected by the outer membrane, which most beta-lactams cross by diffusion through aqueous pores composed of 'porin' proteins. In enterobacteria, a clear correlation exists between porin quantity and cephalosporin resistance, suggesting that the outer membrane is the sole barrier to drug entry. Such relationships are less clear for Pseudomonas aeruginosa, where the cell may contain additional barriers between the outer membrane and the PBPs. Although elevated cephalosporin resistance often is attributed to a single factor (PBP-modification, beta-lactamase action or impermeability) an organism's response to a drug often reflects the interplay of several factors. Mathematical models can be proposed to describe this interplay.

The Cefoperazone–Sulbactam Combination: In Vitro Qualities Including Beta-Lactamase Stability, Antimicrobial Activity, and Interpretive Criteria for Disk Diffusion Tests

Three concentrations of the penicillanic acid sulfone, sulbactam were tested in combination with cefoperazone against 632 recent clinical bacterial isolates. Cefoperazone was effective alone (less than or equal to 16 micrograms/mL) against 95% of Enterobacteriaceae and combined with 4 micrograms/mL sulbactam inhibited 99.5% of strains. This coverage of enteric bacilli was superior to timentin (99.1%), ceftazidime (98.2%), and tobramycin (90.9%). The minimum inhibitory concentrations (MICs) of cefoperazone-susceptible strains also were markedly decreased by sulbactam (overall MIC90s, 8.0 micrograms/mL for cefoperazone and 1.0 microgram/mL for cefoperazone and 4.0 micrograms/mL for sulbactam). Sulbactam also expanded the spectrum of cefoperazone against Acinetobacter species, some rare Pseudomonas species, and Bacteroides fragilis group species. Sulbactam had direct antimicrobial activity against the acinetobacters and Pseudomonas acidovorans, but the increased activity of cefoperazone-sulbactam against some other Pseudomonas species and anaerobes was attributed to beta-lactamase inhibition. The cefoperazone MICs against beta-lactamase producing Staphylococcus species also were lowered to the level of enzyme-deficient strains. Cefoperazone bactericidal activity was improved by 4.0 micrograms/mL sulbactam, and no antagonism was observed. beta-lactamase hydrolysis studies confirmed a slow hydrolysis of cefoperazone only by TEM beta-lactamases and a high-grade resistance to enzyme breakdown by sulbactam. Differential beta-lactamase affinity studies for cefoperazone and sulbactam showed potential efficacy and applications to plasmid-mediated TEM and OXA enzymes and only marginal effective sulbactam inhibition of Pseudomonas and Klebsiella species enzymes. Disk diffusion studies on 556 strains confirmed the applicability of the cefoperazone 75-micrograms disk to testing routine isolates other than enterococci and methicillin-resistant Staphylococcus aureus. The addition of 4.0 micrograms sulbactam/mL in a fixed concentration to dilution test systems and 15 micrograms sulbactam to the 75 micrograms cefoperazone disk were recommended for in vitro tests. Susceptibility and resistant interpretive criteria for the disk and dilution tests can be applied with confidence. Only 0.4% false-susceptibility errors and a 97.5% absolute interpretive agreement were achieved using the 75 micrograms cefoperazone/15 micrograms sulbactam disk.

In vitro activity of imipenem--a review.

A review is given of the microbiological properties of imipenem, a new carbapenem antibiotic with an exceptionally broad spectrum of antibacterial activity. An evaluation of results of numerous in vitro studies reveals that imipenem effectively inhibited growth of 53 of 55 bacterial species, the mean MIC90 being less than 8 mg/l. The MIC90 for cocci, with the exception of Staphylococcus epidermidis, is in the range of 0.01-3.1 mg/l. The MIC90 for all Enterobacteriaceae is equal to or less than 8 mg/l. Pseudomonas aeruginosa and other non-fermentative gram-negative bacteria are generally susceptible to imipenem, only Pseudomonas maltophilia and Pseudomonas cepacia showing intrinsic resistance. Imipenem is currently the most active drug available against anaerobic bacteria, the MIC usually being below 1 mg/l even for Bacteroides fragilis. Rare bacteria such as Nocardia asteroides, Listeria monocytogenes or fast growing Mycobacterium spp. which cause difficult-to-treat infections are also susceptible to imipenem. Increases in inoculum size have only a minimal effect on activity of the drug. In most species the MBC only slightly exceeded the MIC; however in the case of Streptococcus faecalis the MBC value was many times the MIC value. Synergism has been observed in combinations of imipenem with aminoglycosides, and antagonism in combinations with other beta-lactam antibiotics against Pseudomonas aeruginosa and Serratia marcescens. Imipenem is stable in the presence of the common chromosomal and plasmid-mediated enzymes. Induction of inactivating enzymes was observed in staphylococci, Pseudomonas aeruginosa and Serratia marcescens.

Current mechanisms of resistance to antimicrobial agents in microorganisms causing infection in the patient at risk for infection

The mechanisms of resistance encountered in bacteria causing infection in the patient at risk for infection are diverse. Most resistance currently seen is the result of plasmid transfer rather than mutational events. However, extensive use of antimicrobial agents in the hospital has caused the selection of organisms resistant to many agents by virtue of chromosomally mediated mechanisms. Staphylococcus aureus resistant to beta-lactams due to altered penicillin-binding proteins has become a problem in certain patients such as narcotic addicts and chronic care facility patients exposed to many beta-lactam antibiotics. S. epidermidis has also proved to be a problem in patients with indwelling foreign devices, and altered penicillin-binding proteins also make these organisms resistant to available penicillins and cephalosporins. Streptococcus fecalis has become increasingly resistant to aminoglycosides, erythromycin, and tetracyclines due to plasmid-mediated enzymes. Hemophilus influenzae resistant to both penicillins and chloramphenicol by virtue of beta-lactamases and chloramphenicol transacetylase has been encountered. Beta-lactamase-mediated resistance of Enterobacteriaceae, Escherichia coli, and Klebsiella pneumoniae to betalactam antibiotics has increased, and resistance of Serratia marcescens and Pseudomonas aeruginosa to aminoglycosides and penicillins is a widespread phenomenon. Mechanisms to reduce resistance will include not only careful attention to hygienic practices but also more appropriate use of antibiotics selecting the proper agent depending on the type of patient and environment in which the infection develops.

Influence of methoxy-substitution of β-lactam compounds on the interaction with various β-lactamases

The interaction of 6 alpha-(temocillin) and 7 alpha-methoxy substituted (cefoxitin) beta-lactam compounds with various beta-lactamases was studied employing enzyme kinetics and compared to that of unsubstituted compounds. Both chromosomally mediated enzymes from Enterobacter cloacae and Citrobacter freundii were competitively inhibited by the methoxy-substituted compounds. Higher concentrations of cefoxitin caused a competitive inhibition of the plasmid-mediated Tem-1 enzyme, whereas temocillin led to a non-competitive inhibition of the Tem-1 enzyme. These results indicate that the discrepancies in the interaction on the above mentioned compounds have to be attributed to the different molecular structure of the beta-lactam nucleus. Moreover, no predictions can be made on the basis of an analogy between 6 alpha-methoxy-penams and 7 alpha-methoxy cephems.

Interaction of New Cepbalosporins with β-Lactamases and β-Lactamase-Producing Gram-Negative Bacilli

Resistance to aminoglycosides and β-lactam antibiotics among gram-negative bacteria that cause nosocomial infections has become a serious problem in medical centers around the world. Resistance to these drugs commonly involves chromosomally or plasmid-mediated enzymes that modify or inactivate the drug. Recent developments in research on β-lactam antibiotics have resulted in compounds with increased stability to enzymatic inactivation and, in addition, with potent activity against many aerobic gram-negative organisms, including Pseudomonas. This study examines some of these new compounds, including cefoperazone and the aminothiazole oximes cefotaxime, ceftazidime, and ceftizoxime, in relation to their stability to cell-free β-lactamase preparations, activity against β-lactamase-producing strains, and susceptibility to increases in inoculum size. Overall, the aminothiazole oximes show a high degree of stability to β-lactamases produced by aerobic gram-negative rods, as well as a high order of activity against the producing organisms although all compounds tested are susceptible to inoculum effects. In contrast, cefoperazone is susceptible to β-lactamases, shows weak activity against β-lactamase-producing strains, and is highly susceptible to inoculum effects. None of the compounds tested shows good activity against β-lactamase-producing strains of Bacteroides fragilis.