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.

Evaluation of extracts of the root of Landolphia owerrience for antibacterial activity

A new phenotypic test, called the Carbapenem Inactivation Method (CIM), was developed to detect carbapenemase activity in Gram-negative rods within eight hours. This method showed high concordance with results obtained by PCR to detect genes coding for the carbapenemases KPC, NDM, OXA-48, VIM, IMP and OXA-23. It allows reliable detection of carbapenemase activity encoded by various genes in species of Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae), but also in non-fermenters Pseudomonas aeruginosa and Acinetobacter baumannii. The CIM was shown to be a cost-effective and highly robust phenotypic screening method that can reliably detect carbapenemase activity.

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The carbapenem inactivation method (CIM), a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in gram-negative rods.

Klebsiella pneumoniae isolates from 11 patients at the Miriam Hospital were identified as resistant to cefoxitin and ceftibuten as well as to aztreonam, cefotaxime, and ceftazidime. Resistance could be transferred by conjugation or transformation with plasmid DNA into Escherichia coli and was due to the production of a beta-lactamase with an isoelectric point of 8.4 named MIR-1. In E. coli, MIR-1 conferred resistance to aztreonam, cefotaxime, ceftazidime, ceftibuten, ceftriaxone, and such alpha-methoxy beta-lactams as cefmetazole, cefotetan, cefoxitin, and moxalactam. In vitro, MIR-1 hydrolyzed cephalothin and cephaloridine much more rapidly than it did penicillin G, ampicillin, or carbenicillin. Cefotaxime was hydrolyzed at 10% the rate of cephaloridine. Cefoxitin inactivation could only be detected by a microbiological test. The inhibition profile of MIR-1 was similar to that of chromosomally mediated class I beta-lactamases. Potassium clavulanate had little effect on cefoxitin or cefibuten resistance and was a poor inhibitor of MIR-1 activity. Cefoxitin or imipenem did not induce MIR-1. The gene determining MIR-1 was cloned on a 1.4-kb AccI-PstI fragment. Under stringent conditions, probes for TEM-1 and SHV-1 genes and the E. coli ampC gene failed to hybridize with the MIR-1 gene. However, a provisional sequence of 150 bp of the MIR-1 gene proved to be 90% identical to the sequence of ampC from Enterobacter cloacae but only 71% identical to that of E. coli, thus explaining the lack of hybridization to the E. coli ampC probe. Plasmid profiles of the 11 K. pneumoniae clinical isolates were not identical, but each contained a plasmid from 40 to 60 kb that hybridized with the cloned MIR-1 gene. Both transfer-proficient and transfer-deficient MIR-1 plasmids belonged to the N incompatibility group. Thus, the resistance of these K. pneumoniae strains was the result of plasmid acquisition of a class I beta-lactamase, a new resistance determinant that expands the kinds of beta-lactam resistance capable of spread by plasmid dissemination among clinical isolates.

Novel plasmid-mediated beta-lactamase (MIR-1) conferring resistance to oxyimino- and alpha-methoxy beta-lactams in clinical isolates of Klebsiella pneumoniae.

From February to November 1997, 29 inpatients at Ramon y Cajal Hospital, Madrid, Spain, were determined to be either colonized or infected with imipenem- and meropenem-resistant Acinetobacter baumannii (IMRAB) strains (MICs, 128 to 256 microg/ml). A wide antibiotic multiresistance profile was observed with IMRAB strains. For typing IMRAB isolates, pulsed-field gel electrophoresis was used. For comparative purposes, 30 imipenem- and meropenem-susceptible A. baumannii (IMSAB) strains isolated before, during, and after the outbreak were included in this study. The molecular-typing results showed that the outbreak was caused by a single IMRAB strain (genotype A). By cloning experiments we identified a class D beta-lactamase (OXA-24) encoded in the chromosomal DNA of this IMRAB strain which showed carbapenem hydrolysis. Moreover, the outer membrane profile of the IMRAB strain showed a reduction in the expression of two porins at 22 and 33 kDa when compared with genetically related IMSAB isolates. In addition no efflux mechanisms were identified in the IMRAB strains. In summary, we report here the molecular characterization of a nosocomial outbreak caused by one multiresistant A. baumannii epidemic strain that harbors a carbapenem-hydrolyzing enzyme. Although alterations in the penicillin-binding proteins cannot be ruled out, the reduction in the expression of two porins and the presence of this OXA-derived beta-lactamase are involved in the carbapenem resistance of the epidemic nosocomial IMRAB strain.

Characterization of a nosocomial outbreak caused by a multiresistant Acinetobacter baumannii strain with a carbapenem-hydrolyzing enzyme: high-level carbapenem resistance in A. baumannii is not due solely to the presence of beta-lactamases.

Detection of beta-lactamase production by gram-negative bacteria.

A novel and time-saving method for assessing bactericidal activities of beta-lactam antibiotics on agar plates is described. Minimal inhibitory concentrations (MICs) were determined by the agar dilution method. A potent beta-lactamase solution was sprayed onto the plates to inactivate the antibiotic. After further incubation at 37 degrees C overnight, the minimal concentration at which no visible growth occurred on the plates was defined as minimal bactericidal concentration (MBC). With undiluted culture as the inoculum, strains of Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae showed a marked increase in MBC values compared with the values of MIC. There was a marked decrease in both the MICs and MBCs with diminution of inoculum size. The two concentrations were almost the same when the inoculum size was decreased to a 10(-4) dilution. In contrast, MIC and MBC for enterococci showed no marked decrease with decrease in inoculum size. Although the present study was performed with beta-lactamase-unstable penicillins and cephalosporins, the method can be applicable with any beta-lactam antibiotic if optimal and potent enzymes are available.

Bacteriostatic and Bactericidal Activities of Selected Beta-Lactam Antibiotics Studied on Agar Plates

Biological β-lactam antibiotic-inactivating activities were detected in bacteriuria and suppurating pleural fluids. Clinical specimens were sterilized with membrane filters and the amounts of ampicillin and/or cephalothin which were being inactivated by 1 ml of each filtrate were determined. In general, filtrates which originally yielded Klebsiella sp. tended to show activity against ampicillin; whereas those yielding Enterobacter sp. and Pseudomonas aeruginosa showed activity against cephalothin.

Possible beta-lactamase activities detectable in infective clinical specimens.

Quantitative bactericidal activities of β-lactam antibiotics were determined by the agar plate method. Broth cultures, of which the colony-forming units were counted before the study, were inoculated on antibiotic-containing agar plates, utilizing a 10 −3 , 10 −2 , or 10 −1 dilution or undiluted culture plated with each 0.001-ml calibrated loop. These plates were incubated at 37°C overnight, and the minimal drug concentration at which no bacterial growth was observed on the plates was defined as minimal inhibitory concentration. After this procedure, the agar surface was treated with β-lactamase spray to inactivate the antibiotic. These plates were incubated again at 37°C overnight. The minimal drug concentration at which no evidence of bacterial growth was visible on the plates (resulting in a 100% kill) was defined as minimal bactericidal concentration. The lowest concentration which reduced the number of colony-forming units to 1/1,000 that in the original inoculum (resulting in a 99.9% kill) was defined as minimal lethal concentration. When compared for Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis , alpha-hemolytic streptococcus (non-enterococcal), beta-hemolytic streptococcus, and enterococcus, the minimal bactericidal concentrations were generally several fold higher than the minimal inhibitory concentrations. Minimal lethal concentrations were virtually the same as minimal inhibitory concentrations for gram-negative strains; however, for some gram-positive strains, minimal lethal concentrations were higher than minimal inhibitory concentrations.

Quantitative Assessment of Bactericidal Activities of β-Lactam Antibiotics by Agar Plate Method

Comparative bactericidal activities were determined utilizing a relatively large number of test strains, in both agar and broth media, with special reference to the time of exposure of the bacteria to certain β-lactam antibiotics. It was apparent that the activities increase with time. The concentrations producing a 99.9% kill with cephalothin for Escherichia coli, Klebsiella sp., and carbenicillin for Pseudomonas aeruginosa were higher in broth than in agar. In contrast, those of benzylpenicillin for α-streptococcus( non-enterococcal) were higher in agar than in broth. If the bactericidal concentrations with 3-hour or 6-hour exposuret o antibiotics were used as the criterion, these concentrations of carbenicillin for P. aeruginosa, and benzylpenicillin for α-streptococcus were, in particular, unusually high compared with the conventionally determined bacteriostatic concentrations( MICs).

Gohta Masuda

Papers

Detection of beta-lactamase production by gram-negative bacteria.

Bacteriostatic and Bactericidal Activities of Selected Beta-Lactam Antibiotics Studied on Agar Plates

Possible beta-lactamase activities detectable in infective clinical specimens.

Quantitative Assessment of Bactericidal Activities of β-Lactam Antibiotics by Agar Plate Method

Comparative bactericidal activities of beta-lactam antibiotics determined in agar and broth media.