Maria F. Mojica

Cross-class metallo-β-lactamase inhibition by bisthiazolidines reveals multiple binding modes

Significance Bacterial diseases remain a huge burden on healthcare worldwide, with the emergence and re-emergence of strains resistant to currently used antibiotics posing an increasing clinical threat. Metallo-β-lactamases (MBLs) are key determinants of antibiotic resistance because they hydrolyze almost all β-lactam antibiotics and are unaffected by currently available β-lactamase inhibitors (βLIs). The structural diversity between MBLs has proved problematic when designing βLIs effective against all MBL targets. Here we show a series of small compounds, bisthiazolidines, which act as inhibitors of all MBL types, restoring the efficacy of currently used antibiotics against resistant bacterial strains producing different MBLs. High-resolution crystal structures reveal how diverse MBLs are inhibited by the unexpected versatility of bisthiazolidine binding, raising implications for future βLI design. Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics and are unaffected by clinically available β-lactamase inhibitors (βLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of βLIs effective against all enzymes. Bisthiazolidines (BTZs) are carboxylate-containing, bicyclic compounds, considered as penicillin analogs with an additional free thiol. Here, we show both l- and d-BTZ enantiomers are micromolar competitive βLIs of all MBL classes in vitro, with Kis of 6–15 µM or 36–84 µM for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10–12 µM for the B3 enzyme L1. Against the B2 MBL Sfh-I, the l-BTZ enantiomers exhibit 100-fold lower Kis (0.26–0.36 µM) than d-BTZs (26–29 µM). Importantly, cell-based time-kill assays show BTZs restore β-lactam susceptibility of Escherichia coli-producing MBLs (IMP-1, Sfh-1, BcII, and GOB-18) and, significantly, an extensively drug-resistant Stenotrophomonas maltophilia clinical isolate expressing L1. BTZs therefore inhibit the full range of MBLs and potentiate β-lactam activity against producer pathogens. X-ray crystal structures reveal insights into diverse BTZ binding modes, varying with orientation of the carboxylate and thiol moieties. BTZs bind the di-zinc centers of B1 (IMP-1; BcII) and B3 (L1) MBLs via the free thiol, but orient differently depending upon stereochemistry. In contrast, the l-BTZ carboxylate dominates interactions with the monozinc B2 MBL Sfh-I, with the thiol uninvolved. d-BTZ complexes most closely resemble β-lactam binding to B1 MBLs, but feature an unprecedented disruption of the D120–zinc interaction. Cross-class MBL inhibition therefore arises from the unexpected versatility of BTZ binding.

B1-metallo- β-lactamases: Where do we stand?

Metallo-β-Lactamases (MBLs) are class Bβ-lactamases that hydrolyze almost all clinically-availableβ-lactam antibiotics. MBLs feature the distinctive αβ/βα sandwich fold of the metallo-hydrolase/oxidoreductase superfamily and possess a shallow active-site groove containing one or two divalent zinc ions, flanked by flexible loops. According to sequence identity and zinc ion dependence, MBLs are classified into three subclasses (B1, B2 and B3), of which the B1 subclass enzymes have emerged as the most clinically significant. Differences among the active site architectures, the nature of zinc ligands, and the catalytic mechanisms have limited the development of a common inhibitor. In this review, we will describe the molecular epidemiology and structural studies of the most prominent representatives of class B1 MBLs (NDM-1, IMP-1 and VIM-2) and describe the implications for inhibitor design to counter this growing clinical threat.

Carbapenem-resistant Enterobacter cloacae isolates producing KPC-3, North Dakota, USA.

To the Editor: Carbapenem-resistant Enterobacteriaceae (CRE) continue to emerge as a serious public health threat throughout the world (1). CRE infections in the United States are often mediated by acquisition of Klebsiella pneumoniae carbapenemase (KPC) expressed by Klebsiella spp., although KPC is also found in other genera (2). The spread of KPC-producing, gram-negative bacteria in hospitals has been linked to severity of illness, co-existing medical conditions, exposure to antimicrobial drugs, and need for chronic care (3). After reporting of CRE infections to the North Dakota Department of Health became mandatory in 2011, a total of 20 CRE cases were noted in 12 of 53 counties (2.9 cases/100,000 population [4]). Most cases involved infection with Enterobacter cloacae and occurred in Cass County, where the state’s largest city, Fargo, is located. We describe an outbreak of clonal carbapenem-resistant E. cloacae in a health care system in Fargo. Sanford Health is a 583-bed, acute-care facility, representing ≈70% of acute-care beds in Fargo. The hospital handles >27,000 admissions/year and serves as a referral center for a large area of the state, and the only long-term acute-care (LTAC) facility in the eastern half of the state operates on its campus. During December 2011–December 2012, all isolates of Enterobacteriaceae with reduced susceptibility to ertapenem (MIC ≥1 µg/mL) identified at the hospital’s clinical microbiology laboratory were screened for carbapenemase production by using the modified Hodge test (mHT), according to Clinical and Laboratory Standards Institute recommendations (5). Identification and susceptibility testing were done with the MicroScan system (Siemens Healthcare Diagnostics, Tarrytown, NY, USA); MICs of carbapenems were confirmed with Etest (bioMerieux, Durham, NC, USA). Three carbapenem-resistant E. cloacae isolates from documented cases of CRE infection at the hospital during 2010 were analyzed for comparison. To characterize carbapenem-resistant and mHT-positive isolates, we used PCR to amplify and sequence the carbapenemase genes blaIMP, blaNDM, blaVIM, and blaKPC by using established methods (6). The upstream sequence of blaKPC-positive strains was analyzed to determine the isoform of the transposon Tn4401 that harbored blaKPC (7). We investigated genetic similarity among isolates by repetitive sequence-based PCR; isolates with >95% similarity were considered clonal (6). We also sequenced the highly conserved hsp60 gene (8) and attempted conjugative transfer of the blaKPC gene by growing KPC-producing E. cloacae along with sodium azide–resistant Escherichia coli J-53. As part of the study, we examined records of patients from whom carbapenem-resistant E. cloacae was isolated. The study was approved by the Institutional Review Board at Sanford Health. During December 2011–December 2012, a total of 19 single-patient E. cloacae isolates and 1 E. aerogenes isolate had positive mHT results. blaKPC was detected in 17 of the 19 E. cloacae isolates and in the 3 carbapenem-resistant E. cloacae isolates from 2010. For all 20 of those isolates, sequencing revealed blaKPC-3 in association with isoform d of the transposon Tn4401, and all isolates were clonally related (Figure). All 20 isolates also had an identical hsp60 sequence belonging to cluster VI in the Hoffman and Roggenkamp scheme (8). Conjugation of a blaKPC-containing plasmid into E. coli J-53 was successful for 1 strain. Figure Genetic typing of carbapenem-resistant Enterobacter cloacae identified from patients at Sanford Health in Fargo, North Dakota, USA. Repetitive sequence–based PCR was used. The dendrogram at left displays the percentage similarity among band patterns ... All 20 of the patients from whom KPC-producing CRE isolates were obtained (17 from this study, 3 from 2010) had been hospitalized at Sanford Health during the 3 months before CRE isolation; 13 (65%) were admitted to intensive care. In addition, 13 (65%) patients had been admitted to the LTAC during the year before CRE isolation. Co-colonization with multidrug-resistant bacteria was documented in 16 (80%) patients, including extended-spectrum β-lactamase–producing and carbapenem-resistant organisms in 4 and 2 patients, respectively. Seven (35%) patients died; 3 (15%) deaths were attributed to CRE infection. One of the patients was a neonate 30 days of age. The finding of KPC-3–producing E. cloacae in North Dakota contrasts with the predominant epidemiology of CRE across the United States. Most CRE cases nationwide are caused by KPC-producing K. pneumoniae (2). KPC-type β-lactamases were previously identified in diverse strains of Enterobacter spp. from an urban health care system in Detroit, accounting for ≈15% of CRE (9). In contrast, our genetic analysis reveals a uniform genetic background among KPC-producing E. cloacae, which suggests horizontal dissemination of an outbreak strain. Because active surveillance programs do not exist at our facility, this study probably underestimates the extent of CRE spread. We found that patients with KPC-producing E. cloacae in this sample were exposed to an LTAC and concomitantly were colonized or infected with other multidrug-resistant organisms (9). Although the spatio-temporal origin of the outbreak (acute care vs. LTAC) remains undefined, these findings likely reflect longer exposure to the continuum of care and higher rates of co-existing conditions within the LTAC population. This outbreak of KPC-producing E. cloacae infections in a health care system in North Dakota highlights the infection control challenges of long-term care facilities and the potential role they play in CRE dissemination.

Successful Treatment of Bloodstream Infection Due to Metallo-β-Lactamase-Producing Stenotrophomonas maltophilia in a Renal Transplant Patient

ABSTRACT Stenotrophomonas maltophilia is an emerging multidrug-resistant (MDR) opportunistic pathogen for which new antibiotic options are urgently needed. We report our clinical experience treating a 19-year-old renal transplant recipient who developed prolonged bacteremia due to metallo-β-lactamase-producing S. maltophilia refractory to conventional treatment. The infection recurred despite a prolonged course of colistimethate sodium (colistin) but resolved with the use of a novel drug combination with clinical efficacy against the patients S. maltophilia isolate.

Avibactam Restores the Susceptibility of Clinical Isolates of Stenotrophomonas maltophilia to Aztreonam

ABSTRACT Stenotrophomonas maltophilia is an emerging opportunistic pathogen, classified by the World Health Organization as one of the leading multidrug-resistant organisms in hospital settings. The need to discover novel compounds and/or combination therapies for S. maltophilia is urgent. We demonstrate the in vitro efficacy of aztreonam-avibactam (ATM-AVI) against S. maltophilia and kinetically characterize the inhibition of the L2 β-lactamase by avibactam. ATM-AVI overcomes aztreonam resistance in selected clinical strains of S. maltophilia, addressing an unmet medical need.

Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase

ABSTRACT PER β-lactamases are an emerging family of extended-spectrum β-lactamases (ESBL) found in Gram-negative bacteria. PER β-lactamases are unique among class A enzymes as they possess an inverted omega (Ω) loop and extended B3 β-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-β-lactam β-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k2/K = 2 × 103 ± 0.1 × 103 M−1 s−1, where k2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D β-lactamases (i.e., k2/K range of ≈103 M−1 s−1) and not class A β-lactamases (i.e., k2/K range, 104 to 105 M−1 s−1). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] → 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the β-lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k2/K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 β-lactamase, we tested different β-lactam–AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing blaPER-2. Our studies define the inactivation of the PER-2 ESBL by AVI and suggest that the biophysical properties of the active site contribute to determining the efficiency of inactivation.

First Report of a Verona Integron-Encoded Metallo-β-Lactamase-Producing Klebsiella pneumoniae Infection in a Child in the United States

We report the first case of a child in the United States infected with an organism producing a Verona Integron-Encoded Metallo-β-Lactamase. This child succumbed to a ventilator-associated pneumonia caused by a Klebsiella pneumoniae producing this resistance mechanism.