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.

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.

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.

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 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.

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.

What we may expect from novel antibacterial agents in the pipeline with respect to resistance and pharmacodynamic principles.

There are some 43 small molecules in the antibiotic development pipeline from late preclinical stage (7 compounds) through Phase 1 (11 molecules), Phase 2 (13 molecules) to Phase 3 (12 molecules). The majority of these are representatives of established antibiotic classes that have been modified to address problems of resistance. In addition, there is considerable activity around the discovery of novel classes of ß-lactamase inhibitors with 10 combinations representing 4 inhibitor classes, at different stages of development. The combination of such inhibitors, which have broad activity against serine ß-lactamases and may even inhibit some penicillin binding proteins, with carbapenems, cephalosporins or aztreonam, provides enhanced activity against multi-drug resistant Gram-negative bacteria. There are 6 molecules representing novel classes of antibiotics but only one of these, murepavadin, is expected to have activity against a Gram-negative pathogenic bacterium (Pseudomonas aeruginosa). Although the new analogues of existing classes, and novel combinations, have been designed to address specific resistance problems, it is by no means certain than they will not be affected by the general mechanisms of resistance, particularly decreased net flux across the Gram-negative outer membrane. The potential impact of resistance mechanisms on the new agents is assessed and the ways in which PK/PD studies are used to design dosing regimens for the new agents, especially combinations, as well as to improve dosing of existing antibiotics are discussed.

Epidemiological expansion, structural studies, and clinical challenges of new ß-lactamases from gram-negative bacteria.

ß-Lactamase evolution presents to the infectious disease community a major challenge in the treatment of infections caused by multidrug-resistant gram-negative bacteria. Because over 1,000 of these naturally occurring ß-lactamases exist, attempts to correlate structure and function have become daunting. Although new enzymes in the extended-spectrum ß-lactamase (ESBL) families are frequently identified, the older CTX-M-14 and CTX-M-15 enzymes have become the most prevalent ESBLs in global surveillance. Carbapenemases with either serine-based or zinc-facilitated hydrolysis mechanisms are posing some of the most critical problems. Most geographical regions now report KPC serine carbapenemases and the metallo-ß-lactamases VIM, IMP, and NDM-1, even though NDM-1 was only recently identified. The rapid emergence of these newer enzymes, with multiple ß-lactamases appearing in a single organism, makes the design of new ß-lactamase inactivators or ß-lactamase-stable ß-lactams all the more difficult. Combination therapy will likely be required to counteract the continuing evolution of these insidious enzymes in multidrug-resistant pathogens.

In vitro susceptibility of characterized ß-lactamase-producing strains tested with avibactam combinations.

Avibactam, a broad-spectrum ß-lactamase inhibitor, was tested with ceftazidime, ceftaroline, or aztreonam against 57 well-characterized Gram-negative strains producing ß-lactamases from all molecular classes. Most strains were nonsusceptible to the ß-lactams alone. Against AmpC-, extended-spectrum ß-lactamase (ESBL)-, and KPC-producing Enterobacteriaceae or Pseudomonas aeruginosa, avibactam lowered ceftazidime, ceftaroline, or aztreonam MICs up to 2,048-fold, to ≤4 µg/ml. Aztreonam-avibactam MICs against a VIM-1 metallo-ß-lactamase-producing Enterobacter cloacae and a VIM-1/KPC-3-producing Escherichia coli isolate were 0.12 and 8 µg/ml, respectively.

Investigational antimicrobial agents of 2013.

New antimicrobial agents are always needed to counteract the resistant pathogens that continue to be selected by current therapeutic regimens. This review provides a survey of known antimicrobial agents that were currently in clinical development in the fall of 2012 and spring of 2013. Data were collected from published literature primarily from 2010 to 2012, meeting abstracts (2011 to 2012), government websites, and company websites when appropriate. Compared to what was reported in previous surveys, a surprising number of new agents are currently in company pipelines, particularly in phase 3 clinical development. Familiar antibacterial classes of the quinolones, tetracyclines, oxazolidinones, glycopeptides, and cephalosporins are represented by entities with enhanced antimicrobial or pharmacological properties. More importantly, compounds of novel chemical structures targeting bacterial pathways not previously exploited are under development. Some of the most promising compounds include novel ß-lactamase inhibitor combinations that target many multidrug-resistant Gram-negative bacteria, a critical medical need. Although new antimicrobial agents will continue to be needed to address increasing antibiotic resistance, there are novel agents in development to tackle at least some of the more worrisome pathogens in the current nosocomial setting.

In vitro activity of ceftolozane-tazobactam as determined by broth dilution and agar diffusion assays against recent U.S. Escherichia coli isolates from 2010 to 2011 carrying CTX-M-type extended-spectrum ß-lactamases.

Ceftolozane MIC(50)/MIC(90)s were 4/8 µg/ml when tested against 26 CTX-M-14-type-producing isolates and 64/>64 µg/ml against 219 CTX-M-15-type-producing isolates. The addition of 4 µg/ml tazobactam lowered the ceftolozane MIC50/MIC9(0)s to ≤ 0.25/0.5 µg/ml by broth microdilution and Etest. The zone diameters for the ceftolozane-tazobactam disks were 23 to 29 mm for 92.2% of the isolates.

7-(4-Alkylidenylpiperidinyl)-quinolone bacterial topoisomerase inhibitors.

Novel antibacterial fluoroquinolone agents bearing a 4-alkylidenylpiperidine 7-position substituent are active against quinolone-susceptible and quinolone-resistant gram-positive bacteria, including Streptococcus pneumoniae and MRSA. Analogs 22b, 23c, and 24 demonstrated superior in vitro and in vivo efficacy to ciprofloxacin against these cocci.

Epidemiology and risk factors for isolation of Escherichia coli producing CTX-M-type extended-spectrum ß-lactamase in a large U.S. Medical Center.

A case-case-control study was conducted to identify independent risk factors for recovery of Escherichia coli strains producing CTX-M-type extended-spectrum ß-lactamases (CTX-M E. coli) within a large Southeastern Michigan medical center. Unique cases with isolation of ESBL-producing E. coli from February 2010 through July 2011 were analyzed by PCR for blaCTX-M, blaTEM, and blaSHV genes. Patients with CTX-M E. coli were compared to patients with E. coli strains not producing CTX-M-type ESBLs (non-CTX-M E. coli) and uninfected controls. Of 575 patients with ESBL-producing E. coli, 491 (85.4%) isolates contained a CTX-M ESBL gene. A total of 319 (84.6%) patients with CTX-M E. coli (282 [74.8%] CTX-M-15 type) were compared to 58 (15.4%) non-CTX-M E. coli patients and to uninfected controls. Independent risk factors for CTX-M E. coli isolation compared to non-CTX-M E. coli included male gender, impaired consciousness, H2 blocker use, immunosuppression, and exposure to penicillins and/or trimethoprim-sulfamethoxazole. Compared to uninfected controls, independent risk factors for isolation of CTX-M E. coli included presence of a urinary catheter, previous urinary tract infection, exposure to oxyimino-cephalosporins, dependent functional status, non-home residence, and multiple comorbid conditions. Within 48 h of admission, community-acquired CTX-M E. coli (n = 51 [16%]) and non-CTX-M E coli (n = 11 [19%]) strains were isolated from patients with no recent health care contacts. CTX-M E. coli strains were more resistant to multiple antibiotics than non-CTX-M E. coli strains. CTX-M-encoding genes, especially bla(CTX-M-15) type, represented the most common ESBL determinants from ESBL-producing E. coli, the majority of which were present upon admission. Septic patients with risk factors for isolation of CTX-M E. coli should be empirically treated with appropriate agents. Regional infection control efforts and judicious antibiotic use are needed to control the spread of these organisms.

Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase.

Antimicrobial-modifying resistance enzymes have traditionally been class specific, having coevolved with the antibiotics they inactivate. Fluoroquinolones, antimicrobial agents used extensively in medicine and agriculture, are synthetic and have been considered safe from naturally occurring antimicrobial-modifying enzymes. We describe reduced susceptibility to ciprofloxacin in clinical bacterial isolates conferred by a variant of the gene encoding aminoglycoside acetyltransferase AAC(6')-Ib. This enzyme reduces the activity of ciprofloxacin by N-acetylation at the amino nitrogen on its piperazinyl substituent. Although approximately 30 variants of this gene have been reported since 1986, the two base-pair changes responsible for the ciprofloxacin modification phenotype are unique to this variant, first reported in 2003 and now widely disseminated. An intense increase in the medical use of ciprofloxacin seems to have been accompanied by a notable development: a single-function resistance enzyme has crossed class boundaries, and is now capable of enzymatically undermining two unrelated antimicrobial agents, one of them fully synthetic.

The ABCD's of ß-lactamase nomenclature.

ß-Lactamases can be named on the basis of molecular characteristics or functional properties. Molecular classes A, B, C, and D define an enzyme according to amino acid sequence and conserved motifs. Functional groups 1, 2, and 3 are used to assign a clinically useful description to a family of enzymes, with subgroups designated according to substrate and inhibitor profiles. In addition, other designations are used to define the functionality of specific subgroups, such as extended-spectrum ß-lactamases, or ESBLs, and inhibitor-resistant TEM, or IRT, ß-lactamases. None of these systems provides an unambiguous description of this versatile set of enzymes. A proposed classification system involving microbiological, molecular, and biochemical properties is described, based on the traditional classes A, B, C, and D and functional groups 1, 2, and 3 designations.

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.