beta-Lactamases are the commonest single cause of bacterial resistance to beta-lactam antibiotics. Numerous chromosomal and plasmid-mediated types are known and may be classified by their sequences or phenotypic properties. The ability of a beta-lactamase to cause resistance varies with its activity, quantity, and cellular location and, for gram-negative organisms, the permeability of the producer strain. beta-Lactamases sometimes cause obvious resistance to substrate drugs in routine tests; often, however, these enzymes reduce susceptibility without causing resistance at current, pharmacologically chosen breakpoints. This review considers the ability of the prevalent beta-lactamases to cause resistance to widely used beta-lactams, whether resistance is accurately reflected in routine tests, and the extent to which the antibiogram for an organism can be used to predict the type of beta-lactamase that it produces.

https://courses.washington.edu/pharm522/Week9/0139.pdf

beta-Lactamases in laboratory and clinical resistance.

Summary: Since the introduction of penicillin, β-lactam antibiotics have been the antimicrobial agents of choice. Unfortunately, the efficacy of these life-saving antibiotics is significantly threatened by bacterial β-lactamases. β-Lactamases are now responsible for resistance to penicillins, extended-spectrum cephalosporins, monobactams, and carbapenems. In order to overcome β-lactamase-mediated resistance, β-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) were introduced into clinical practice. These inhibitors greatly enhance the efficacy of their partner β-lactams (amoxicillin, ampicillin, piperacillin, and ticarcillin) in the treatment of serious Enterobacteriaceae and penicillin-resistant staphylococcal infections. However, selective pressure from excess antibiotic use accelerated the emergence of resistance to β-lactam-β-lactamase inhibitor combinations. Furthermore, the prevalence of clinically relevant β-lactamases from other classes that are resistant to inhibition is rapidly increasing. There is an urgent need for effective inhibitors that can restore the activity of β-lactams. Here, we review the catalytic mechanisms of each β-lactamase class. We then discuss approaches for circumventing β-lactamase-mediated resistance, including properties and characteristics of mechanism-based inactivators. We next highlight the mechanisms of action and salient clinical and microbiological features of β-lactamase inhibitors. We also emphasize their therapeutic applications. We close by focusing on novel compounds and the chemical features of these agents that may contribute to a “second generation” of inhibitors. The goal for the next 3 decades will be to design inhibitors that will be effective for more than a single class of β-lactamases.

Three Decades of β-Lactamase Inhibitors

The elaboration of r-lactamase-mediated resistance in bacteria has involved increased prevalence in certain organisms (notably Staphylococclus aiureius in the 1940s) and spread to new hosts (such as Haemophilus influienzae and Neisseria gonorrhoeae in the 1970s and Enterococcusfaecalis in the 1980s) but not fundamental changes in the substrate spectra of the enzymes. Hence, penicillins, cephalosporins, and other ,B-lactam antibiotics which were such poor substrates for hydrolysis as to be virtually resistant to usual amounts of ,B-lactamase could be developed. Some pathogens could develop resistance by mutations allowing so much enzyme to be made that even poor substrates were inactivated or bound, but such resistance was limited to organisms with inducible chromosomal 1-lactamase and was not transmissible (27). Consequently, it came as an unwelcome surprise when Klebsiella isolates with plasmid-mediated resistance to broad-spectrum cephalosporins were found in the Federal isolates (Table 1). The genesis of these plasmid-determined extended-spectrum 1-lactamases and the contribution that they have made to our understanding of 1-lactamase structure-activity relationships are the subjects of this review.

Extended-spectrum beta-lactamases.

A number of ways and means have evolved to provide resistance to eubacteria challenged by β-lactams. This review is focused on pathogens that resist by expressing low-affinity targets for these antibiotics, the penicillin-binding proteins (PBPs). Even within this narrow focus, a great variety of strategies have been uncovered such as the acquisition of an additional low-affinity PBP, the overexpression of an endogenous low-affinity PBP, the alteration of endogenous PBPs by point mutations or homologous recombination or a combination of the above.

Penicillin-Binding Proteins and β-Lactam Resistance

β-Lactamases, the major resistance determinant for β-lactam antibiotics in Gram-negative bacteria, are ancient enzymes whose origins can be traced back millions of years ago. These well-studied enzymes, currently numbering almost 2,800 unique proteins, initially emerged from environmental sources, most likely to protect a producing bacterium from attack by naturally occurring β-lactams. Their ancestors were presumably penicillin-binding proteins that share sequence homology with β-lactamases possessing an active-site serine. Metallo-β-lactamases also exist, with one or two catalytically functional zinc ions. Although penicillinases in Gram-positive bacteria were reported shortly after penicillin was introduced clinically, transmissible β-lactamases that could hydrolyze recently approved cephalosporins, monobactams, and carbapenems later became important in Gram-negative pathogens. Nomenclature is based on one of two major systems. Originally, functional classifications were used, based on substrate and inhibitor profiles. A later scheme classifies β-lactamases according to amino acid sequences, resulting in class A, B, C, and D enzymes. A more recent nomenclature combines the molecular and biochemical classifications into 17 functional groups that describe most β-lactamases. Some of the most problematic enzymes in the clinical community include extended-spectrum β-lactamases (ESBLs) and the serine and metallo-carbapenemases, all of which are at least partially addressed with new β-lactamase inhibitor combinations. New enzyme variants continue to be described, partly because of the ease of obtaining sequence data from whole-genome sequencing studies. Often, these new enzymes are devoid of any phenotypic descriptions, making it more difficult for clinicians and antibiotic researchers to address new challenges that may be posed by unusual β-lactamases.

Past and Present Perspectives on β-Lactamases.

Sir, We report on the frequency of Enterobacteriaceae strains which produce plasmid-mediated beta-lactamases and specify the different types of betalactamases. Six hundred and eighty Enterobacteriaceae were consecutively isolated from human pathological specimens by three clinical bacteriological laboratories in 1981-1982. All strains were identified by standard procedures. The MICs of arnpiciUin, amoxiciUin plus clavulanic acid (ratio 2 : 1) and eephalothin were determined for these strains on Mueller-Hinton agar using an inoculum of about 10 s CFU. According to a procedure published elsewhere (1), nitroeephin was added to disrupted bacteria to detect the presence of enzyme. The hydrolysis spectrum of the strains' overall beta-lactamase activity provided a rough substrate profile. The isoelectric point of the beta-lactamases was studied on poly. acrylamide gels by isoelectrofocusing (2, 3) using sonieated extracts that had been purified by ultrafiltration and concentrated when necessary. The betalactamase types were identified by comparison with standard enzymes (2, 3). Beta.lactamase activity was detected in 588 of the 680 strains: 270 showed cephalosporinase activity, 315 broad-spectrum enzyme activity and three, penieillinase activity. The 92 strains without activity included Salmonella spp. (14) and Proteus mirabilis (73). Three hundred and seventeen strains were susceptible to arnpicfllin; those having cephalosporinase activity alone or none at all were presumed to have only chromosome-mediated beta-lactamase. Three hundred and sixty-three (53.38 % of the total) were ampieillin-resistant (MIC > 16/ag/rnl); this ineluded all strains with broad-spectrum beta-Iactamase activity except one (314), plus 37 with cephalosporinase activity, three with penieillinase and nine without enzymatic activity. The isoeleetric point of the beta4actamases of all ampicillin-resistant strains was determined, in addition to that of four susceptible strains (one Escherichia coli with broad-spectrum activity, one Serratia spp., one Otrobacter spp. and one indole-positive Proteus spp.). Values were obtained for 340 strains (92.64 %); 297 of these showed plasmid-mediated beta-lactamase types and 43 showed only chromosome-mediated enzymes. In agreement with other publications (4, 5), the plasmid-mediated beta-lactamase encountered most frequently was TEM-I (87.54 % of the total strains, including those having one or two enzymes). The second most frequent was SHV-1 (in 14.81% of the total); this frequency is somewhat higher than reported in the literature (4, 5). In 19 strains two different types were identified. Two hundred and twenty-nine strains of Escherichia coli, Salmonella spp., Shighella spp. and Proteus mirabilis showed broad-spectrum beta-lactamase activity and all but one were ampicillin-resistant; 17.8% were resistant (MIC>16/~g/ml) to the combination amoxicillin + clavulanic acid and 30% to cephalothin (MIC > 16/ag/ml). Plasmid-mediated beta-lactamases were identified in 40.83 % of the strains, 97.33 % of which were TEM-1 type. The 57 strains of Klebsiella spp. were resistant to ampicillin, 27.27 % were resistant to amoxicillin + clavulanic acid and 32.8 % to cephalothin; broadspectrum activity was detected in 44 strains. Plasmidmediated beta-lactamases were identified in 73.68 % of these strains, SHV-1 (95.23 %) being the most frequent followed by TEM-1 (30.95 %); two different types were identified in 28.57 % of the strains. The frequency of plasmid-mediated beta-lactamases differs within the genus; whereas only one of 11 Klebsiella oxytoca strains carried plasmid-mediated betalactamases, 41 were observed in 43 Klebsiella pneumoniae strains.

Frequency of plasmid-determined beta-lactamases in 680 consecutively isolated strains ofEnterobacteriaceae

beta-Lactamases from 618 isolates of Klebsiella spp. (487 Klebsiella pneumoniae and 131 Klebsiella oxytoca) were identified by analytical isoelectric focusing. The strains were consecutive isolates from 618 different patients in five hospital centres between 1980 and 1984. Most of the chromosomal beta-lactamases from the K. pneumoniae isolates had a pI of 7.1, and most of the K. oxytoca isolates had chromosomal beta-lactamases of pIs 7.8, 5.3 and 5.5. Plasmid-mediated beta-lactamases were produced by 91.8% of K. pneumoniae isolates and 9.9% of K. oxytoca isolates. The beta-lactamase types identified most frequently were SHV-1 in the former species, and TEM-1 in the latter species. Of the K. pneumoniae isolates, 101 produced both of these plasmid-mediated beta-lactamases, while one strain produced an extended-spectrum SHV-like beta-lactamase. Of the 131 K. oxytoca isolates, 20 were hyper-producers of chromosomal beta-lactamase. The susceptibility of this genus to beta-lactam antibiotics is controlled almost exclusively by beta-lactamases, the production of which may differ from one strain to another; thus the empirical therapeutic use of beta-lactam antibiotics is not recommended for this genus.

A survey of β-lactamases from 618 isolates of Klebsiella spp

A total of 1056 strains of Pseudomonas aeruginosa isolated from human clinical specimens from patients was collected from eight laboratories in order to study the frequency of plasmid-determined beta-lactamase producers and the different enzymes represented. The strains from each laboratory comprised consecutive, non-repeated, clinical isolates. In the 166 strains selected because they were carbenicillin resistant, the isoelectric points of the beta-lactamases were studied by means of analytical isoelectric focusing and the different types of plasmid-determined beta-lactamases identified. Seventy-five of the strains (45.18%) were plasmid-mediated beta-lactamase producers; the frequency varied among laboratories from 0% to 100%. Overall the most frequently identified beta-lactamase type was PSE-1 (49.34%) followed by TEM-1 (37.34%). The remaining carbenicillin-resistant strains did not produce plasmid-determined beta-lactamases.

Incidence of strains producing plasmid determined β-lactamases among carbenicillin resistant Pseudomonas aeruginosa

We studied the susceptibility to the amoxycillin/clavulanate combination (2:1), and the characteristics of the beta-lactamases, of 1410 amoxycillin-resistant strains of Enterobacteriaceae (785 of Escherichia coli and 625 of Klebsiella spp.). Of the E. coli strains, 97.07% were susceptible to the combination and 98.85% produced plasmid-mediated beta-lactamases. Of the Klebsiella spp. strains, 87.84% were susceptible to the combination and 75.36% produced plasmid-mediated beta-lactamases. In both cases, the differences in MIC values, according to the characteristics of the beta-lactamases of the strains, were statistically significant. Of the 99 strains of E. coli and Klebsiella spp. that were not inhibited by amoxycillin 16 mg/l with clavulanate 8 mg/l, 19 hyperproduced chromosomal beta-lactamase; five produced a plasmid-type beta-lactamase as well. Fifty-seven strains produced two plasmid-type beta-lactamases.

M. Hermida

Papers

Frequency of plasmid-determined beta-lactamases in 680 consecutively isolated strains ofEnterobacteriaceae

A survey of β-lactamases from 618 isolates of Klebsiella spp

Incidence of strains producing plasmid determined β-lactamases among carbenicillin resistant Pseudomonas aeruginosa

Activity of amoxycillin/clavulanate against β-lactamase-producing Escherichia coli and Klebsiella spp.

β-Lactamases and snsceptibility phenotypes to β-lactam antibiotics in Escherichia coli strains