Classification for ß-lactamases: historical perspectives.

INTRODUCTION: ß-Lactamases are some of the most prevalent and well-studied families of enzymes, especially in the area of antibiotic resistance. Early attempts to categorize them used either functional names, such as penicillinase or cephalosporinase or structural designations into classes A and B. Increasing diversity of the properties of these enzymes has required a more expansive approach to nomenclature. AREAS COVERED: Historical designations for early ß-lactamases relied heavily on functional names based on the biochemical properties of purified enzymes. As amino acid sequences began to be reported for a select group of these enzymes, classes of ß-lactamases were defined, with a major lumping of enzymes into those that had active site serine residues (class A, C, and D) and those that were metallo-ß-lactamases (MBLs or class B). More recent classification schemes, as determined through a Medline search, have attempted to incorporate both functional and structural features, using functional groups and subgroups to name ß-lactamases within the same structural class. Nomenclature of these enzymes is now under the purview of the NCBI (National Center for Biotechnology Information). EXPERT OPINION: ß-Lactamase nomenclature will continue to evolve with the identification of new enzymes and new functionalities.

Consensus on ß-Lactamase Nomenclature.

Assigning names to ß-lactamase variants has been inconsistent and has led to confusion in the published literature. The common availability of whole genome sequencing has resulted in an exponential growth in the number of new ß-lactamase genes. In November 2021 an international group of ß-lactamase experts met virtually to develop a consensus for the way naturally-occurring ß-lactamase genes should be named. This document formalizes the process for naming novel ß-lactamases, followed by their subsequent publication.

In vitro antibacterial activity of cefiderocol against recent multidrug-resistant carbapenem-nonsusceptible Enterobacterales isolates.

Cefiderocol, a siderophore-containing cephalosporin with broad-spectrum antimicrobial activity against many ß-lactam-resistant Gram-negative bacteria, was tested by broth microdilution against 104 carbapenem-non-susceptible Enterobacterales clinical isolates from 2011 to 2018. Carbapenemase identification was determined using PCR followed by targeted gene sequencing or whole genome sequencing (WGS). All isolates were multidrug-resistant, 89.4% (93/104) and produced a serine (KPC or SME) carbapenemase, with as many as four ß-lactamases present. A VIM-1 or NDM-1 metallo-ß-lactamase was confirmed in 6.7% of the isolates (N = 5 and 2, respectively). All isolates were susceptible to cefiderocol, unlike the comparator agents. Susceptibility for comparators ranged from 24.0% for meropenem to 91.3%, 92.3% and 96.1% for imipenem-relebactam, ceftazidime-avibactam and meropenem-vaborbactam, respectively; 48.1%, 75.2% and 79.8% of the isolates were susceptible to omadacycline, colistin and eravacycline, respectively. Two isolates with cefiderocol MICs of 2 mg/L had mutations or deletions of the iron transport genes fhuA/E or fepA, as determined by WGS.

Success and Challenges Associated with Large-Scale Collaborative Surveillance for Carbapenemase Genes in Gram-Negative Bacteria.

The emergence and spread of antimicrobial resistance, especially in Gram-negative bacteria, has led to significant morbidity and increased cost of health care. Large surveillance studies such as the one performed by the Antibiotic Resistance Laboratory Network are immensely valuable in understanding the scope of resistance mechanisms, especially among carbapenemase-producing Gram-negative bacteria. However, the routine laboratory detection of carbapenemases in these bacteria remains challenging and requires further optimization.

Critical analysis of antibacterial agents in clinical development.

The antibacterial agents currently in clinical development are predominantly derivatives of well-established antibiotic classes and were selected to address the class-specific resistance mechanisms and determinants that were known at the time of their discovery. Many of these agents aim to target the antibiotic-resistant priority pathogens listed by the WHO, including Gram-negative bacteria in the critical priority category, such as carbapenem-resistant Acinetobacter, Pseudomonas and Enterobacterales. Although some current compounds in the pipeline have exhibited increased susceptibility rates in surveillance studies that depend on geography, pre-existing cross-resistance both within and across antibacterial classes limits the activity of many of the new agents against the most extensively drug-resistant (XDR) and pan-drug-resistant (PDR) Gram-negative pathogens. In particular, cross-resistance to unrelated classes may occur by co-selection of resistant strains, thus leading to the rapid emergence and subsequent spread of resistance. There is a continued need for innovation and new-class antibacterial agents in order to provide effective therapeutic options against infections specifically caused by XDR and PDR Gram-negative bacteria.

Epidemiology of ß-Lactamase-Producing Pathogens.

ß-Lactam antibiotics have been widely used as therapeutic agents for the past 70 years, resulting in emergence of an abundance of ß-lactam-inactivating ß-lactamases. Although penicillinases in Staphylococcus aureus challenged the initial uses of penicillin, ß-lactamases are most important in Gram-negative bacteria, particularly in enteric and nonfermentative pathogens, where collectively they confer resistance to all ß-lactam-containing antibiotics. Critical ß-lactamases are those enzymes whose genes are encoded on mobile elements that are transferable among species. Major ß-lactamase families include plasmid-mediated extended-spectrum ß-lactamases (ESBLs), AmpC cephalosporinases, and carbapenemases now appearing globally, with geographic preferences for specific variants. CTX-M enzymes include the most common ESBLs that are prevalent in all areas of the world. In contrast, KPC serine carbapenemases are present more frequently in the Americas, the Mediterranean countries, and China, whereas NDM metallo-ß-lactamases are more prevalent in the Indian subcontinent and Eastern Europe. As selective pressure from ß-lactam use continues, multiple ß-lactamases per organism are increasingly common, including pathogens carrying three different carbapenemase genes. These organisms may be spread throughout health care facilities as well as in the community, warranting close attention to increased infection control measures and stewardship of the ß-lactam-containing drugs in an effort to control selection of even more deleterious pathogens.

A Standard Numbering Scheme for Class C ß-Lactamases.

Unlike for classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) ß-lactamases, which complicates communication in the field. Here, we propose a scheme developed through a collaborative approach that considers both sequence and structure, preserves traditional numbering of catalytically important residues (Ser64, Lys67, Tyr150, and Lys315), is adaptable to new variants or enzymes yet to be discovered and includes a variation for genetic and epidemiological applications.

Activity of imipenem/relebactam against carbapenemase-producing Enterobacteriaceae with high colistin resistance.

OBJECTIVES: Imipenem/relebactam, an investigational ß-lactam/ß-lactamase inhibitor combination for treatment of Gram-negative infections, and comparators including ceftazidime/avibactam, piperacillin/tazobactam and colistin were tested for activity against representative carbapenemase-producing Enterobacteriaceae (CPE) isolates. METHODS: MICs of the antimicrobial agents were determined using standard broth microdilution methodology for CPE isolates collected from Indiana patients, primarily during the time frame of 2013-17 (n = 199 of a total of 200 isolates). Inhibitors were tested at 4 mg/L in all combinations. RESULTS: Of the CPE in the study, 199 produced plasmid-encoded KPC class A carbapenemases; 1 Serratia marcescens isolate produced the SME-1 chromosomal class A carbapenemase. MIC50/MIC90 values of imipenem/relebactam were ≤0.25/0.5 mg/L, whereas MIC50/MIC90 values of ceftazidime/avibactam were 1/2 mg/L. Resistance to colistin was observed in 54% (n = 97) of 180 non-Serratia isolates tested (MIC50 of 4 mg/L). Colistin resistance mechanisms included production of a plasmid-encoded mcr-1-like gene (n = 2) or an inactivated mgrB gene. CONCLUSIONS: Imipenem/relebactam was the most potent agent tested against CPE in this study and may be a useful addition to the antimicrobial armamentarium to treat infections caused by these pathogens.

Author Correction: Interplay between ß-lactamases and new ß-lactamase inhibitors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: Interplay between ß-lactamases and new ß-lactamase inhibitors.

In figure 1 of the above article, the structure of ETX2514 was missing a double bond and methyl group. This has now been corrected in the PDF and online. The publisher apologizes to the authors and to the readers for this error.

Interplay between ß-lactamases and new ß-lactamase inhibitors.

Resistance to ß-lactam antibiotics in Gram-negative bacteria is commonly associated with production of ß-lactamases, including extended-spectrum ß-lactamases (ESBLs) and carbapenemases belonging to different molecular classes: those with a catalytically active serine and those with at least one active-site Zn2+ to facilitate hydrolysis. To counteract the hydrolytic activity of these enzymes, combinations of a ß-lactam with a ß-lactamase inhibitor (BLI) have been clinically successful. However, some ß-lactam-BLI combinations have lost their effectiveness against prevalent Gram-negative pathogens that produce ESBLs, carbapenemases or multiple ß-lactamases in the same organism. In this Review, descriptions are provided for medically relevant ß-lactamase families and various BLI combinations that have been developed or are under development. Recently approved inhibitor combinations include the inhibitors avibactam and vaborbactam of the diazabicyclooctanone and boronic acid inhibitor classes, respectively, as new scaffolds for future inhibitor design.

A Meandering Path from Biochemist to Microbiologist.

Not all career paths are clearly defined from the beginning. In my case, biochemistry training in enzymology provided the tools to move from a basic research beginning in biophysical/bioorganic chemistry to an applied microbiology career in antibiotic drug discovery and development. This pathway required essential contributions from family, professional colleagues, co-workers, and mentors who all provided input at critical times during changes in career directions. As a result of applying skills from previous research positions to new sets of challenges, my contributions to various antibiotic research programs led to the introduction of valuable antimicrobial agents currently used in the treatment of infected patients.

Past and Present Perspectives on ß-Lactamases.

ß-Lactamases, the major resistance determinant for ß-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 ß-lactams. Their ancestors were presumably penicillin-binding proteins that share sequence homology with ß-lactamases possessing an active-site serine. Metallo-ß-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 ß-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 ß-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 ß-lactamases. Some of the most problematic enzymes in the clinical community include extended-spectrum ß-lactamases (ESBLs) and the serine and metallo-carbapenemases, all of which are at least partially addressed with new ß-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 ß-lactamases.

Selection of hyperproduction of AmpC and SME-1 in a carbapenem-resistant Serratia marcescens isolate during antibiotic therapy.

Objectives: Antibiotic selective pressure may result in changes to antimicrobial susceptibility throughout the course of infection, especially for organisms that harbour chromosomally encoded AmpC ß-lactamases, notably Enterobacter spp., in which hyperexpression of ampC may be induced following treatment with cephalosporins. In this study, we document a case of bacteraemia caused by a blaSME-1-harbouring Serratia marcescens that subsequently developed resistance to expanded-spectrum cephalosporins, piperacillin/tazobactam and fluoroquinolones, over the course of several months of treatment with piperacillin/tazobactam and ciprofloxacin. Methods: Susceptibility testing and WGS were performed on three S. marcescens isolates from the patient. ß-Lactamase activity in the presence or absence of induction by imipenem was measured by nitrocefin hydrolysis assays. Expression of ampC and blaSME-1 under the same conditions was determined by real-time PCR. Results: WGS demonstrated accumulation of missense and nonsense mutations in ampD associated with stable derepression of AmpC. Gene expression and ß-lactamase activity of both AmpC and SME-1 were inducible in the initial susceptible isolate, but were constitutively high in the resistant isolate, in which total ß-lactamase activity was increased by 128-fold. Conclusions: Although development of such in vitro resistance due to selective pressure imposed by antibiotics is reportedly low in S. marcescens, our findings highlight the need to evaluate isolates on a regular basis during long-term antibiotic therapy.

Unusual carbapenem resistant but ceftriaxone and cefepime susceptible Klebsiella oxytoca isolated from a blood culture: Case report and whole-genome sequencing investigation.

A carbapenem resistant but ceftriaxone and cefepime susceptible Klebsiella oxytoca was isolated from the blood of a patient with polymicrobial bacteremia after 2 weeks of ertapenem treatment. Whole-genome sequencing identified no carbapenemase gene nor plasmid, but only blaOXY-2-8 gene with a mutation in the promoter that's been reported to increase its expression. Two other specific carbapenem resistance mechanisms including mutated porin genes and the AcrAB-TolC efflux system genes were also identified. Clinicians need to be aware of such unusual antibiogram and should not assume carbapenems are always broader spectrum antibiotics than expanded-spectrum cephalosporins.

Game Changers: New ß-Lactamase Inhibitor Combinations Targeting Antibiotic Resistance in Gram-Negative Bacteria.

Recent regulatory approvals for the ß-lactam inhibitor combinations of ceftazidime-avibactam and meropenem-vaborbactam have provided two novel therapeutic options for the treatment of multidrug-resistant infections caused by Gram-negative bacteria. Most importantly, these combination agents have satisfied an important medical need related to antibiotic-resistant Klebsiella pneumoniae that produce serine carbapenemases, especially the Klebsiella pneumoniae carbapenemase (KPC) enzymes. Both combinations contain non-ß-lactam ß-lactamase inhibitors of novel chemical classes not previously developed as antibacterial agents, the diazabicyclooctanes and cyclic boronic acid derivatives. Their rapid development and approval programs have spurred a number of similar inhibitor combinations that will need to differentiate themselves for commercial success. Gaps still exist for the treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa, Acinetobacter spp., and metallo-ß-lactamase-producing pathogens. Overall, the new ß-lactamase inhibitor combinations have infused new life into the search for new antibacterial agents to treat multidrug-resistant bacteria.

In vitro activity of plazomicin against ß-lactamase-producing carbapenem-resistant Enterobacteriaceae (CRE).

Background: Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat because few drugs are available to treat infections caused by these pathogens. Plazomicin is a novel aminoglycoside that recently completed a Phase 3 clinical trial for treatment of infections caused by CRE. Methods: A set of 110 characterized unique CRE patient isolates from central Indiana healthcare centres was tested by microbroth dilution for susceptibility to plazomicin, and to reference aminoglycosides and carbapenems. WGS was conducted to analyse the isolate with an elevated plazomicin MIC. Results: The isolates, 107 of which produced KPC carbapenemases, were 97.3% and 100% non-susceptible to meropenem and imipenem, respectively, with variable rates of non-susceptibility to amikacin (76.4%), gentamicin (18.2%), kanamycin (91.8%) and tobramycin (96.4%). MIC50/MIC90 values for plazomicin were the lowest of all the drugs tested: 0.5/0.5 mg/L for 96 KPC-producing Klebsiella pneumoniae isolates and 0.5/1 mg/L for all 110 carbapenemase-producing isolates. Higher MIC50/MIC90 values were observed for the other antibiotics tested: amikacin (32/32 mg/L), gentamicin (1/16 mg/L), kanamycin (>64/>64 mg/L), tobramycin (32/64 mg/L), imipenem (8/32 mg/L) and meropenem (≥16/≥16 mg/L). Only one isolate, an NDM-1-producing K. pneumoniae strain that carried the armA 16S rRNA methyltransferase gene, was resistant to plazomicin, with an MIC of 256 mg/L; this strain was cross-resistant to all the other antibiotics tested. Conclusions: Plazomicin demonstrated the most potent overall in vitro inhibitory activity of all the aminoglycosides and carbapenems in the study, and has the potential to be an effective agent for the treatment of infections caused by CRE.

Unusual Escherichia coli PBP 3 Insertion Sequence Identified from a Collection of Carbapenem-Resistant Enterobacteriaceae Tested In Vitro with a Combination of Ceftazidime-, Ceftaroline-, or Aztreonam-Avibactam.

Carbapenemase-producing Enterobacteriaceae isolates (n = 110) from health care centers in central Indiana (from 2010 to 2013) were tested for susceptibility to combinations of avibactam (4 µg/ml) with ceftazidime, ceftaroline, or aztreonam. MIC50/MIC90 values were 1/2 µg/ml (ceftazidime-avibactam), 0.5/2 µg/ml (ceftaroline-avibactam), and 0.25/0.5 µg/ml (aztreonam-avibactam.) A ß-lactam MIC of 8 µg/ml was reported for the three combinations against one Escherichia coli isolate with an unusual TIPY insertion following Tyr344 in penicillin-binding protein 3 (PBP 3) as the result of gene duplication.