Rapid Emergence, Subsidence, and Molecular Detection of Escherichia coli Sequence Type 1193-fimH64, a New Disseminated Multidrug-Resistant Commensal and Extraintestinal Pathogen.

Escherichia coli sequence type 1193 (ST1193) is an emerging multidrug-resistant pathogen. We performed longitudinal and cross-sectional surveillance for ST1193 among clinical and fecal E. coli isolates from Minneapolis Veterans Affairs Medical Center (VAMC) patients and their household members, other Minnesota centers, and national VAMCs and compared these ST1193 isolates with archival human and canine ST1193 isolates from Australia (2008). We also developed and extensively validated a novel multiplex PCR assay for ST1193 and its characteristic fimH64 (type 1 fimbrial adhesin) allele. We found that ST1193-H64 (where "H64" refers to a phylogenetic subdivision within ST1193 that is characterized by the fimH64 allele), which was uniformly fluoroquinolone resistant, appeared to emerge in the United States in a geographically staggered fashion beginning around 2011. Its prevalence among clinical and fecal E. coli isolates at the Minneapolis VAMC rose rapidly beginning in 2013, peaked in early 2017, and then plateaued or declined. In comparison with other ST14 complex (STc14) isolates, ST1193-H64 isolates were more extensively multidrug resistant, whereas their virulence genotypes were less extensive but included (uniquely) K1 capsule and fimH64 Pulsed-field gel electrophoresis separated ST1193-H64 isolates from other STc14 isolates and showed genetic commonality between archival Australian versus recent U.S. isolates, fecal versus clinical isolates, and human versus canine isolates. Three main ST1193 pulsotypes differed significantly in resistance profiles and capsular types; emergent pulsotype 2123 was associated with trimethoprim-sulfamethoxazole resistance and K1 (versus K5) capsule. These findings clarify ST1193-H64's temporal prevalence trends as a fluoroquinolone-resistant pathogen and commensal; document clonal subsets with distinctive geographic, temporal, resistance, and virulence gene associations; and establish a new laboratory tool for rapid and simple detection of ST1193-H64.

Variation in resistance traits, phylogenetic backgrounds, and virulence genotypes among Escherichia coli clinical isolates from adjacent hospital campuses serving distinct patient populations.

Escherichia coli sequence type 13 (ST131), an emergent cause of multidrug-resistant extraintestinal infections, has important phylogenetic subsets, notably the H30 and H30Rx subclones, with distinctive resistance profiles and, possibly, clinical associations. To clarify the local prevalence of these ST131 subclones and their associations with antimicrobial resistance, ecological source, and virulence traits, we extensively characterized 233 consecutive E. coli clinical isolates (July and August 2013) from the University of Minnesota Medical Center-Fairview Infectious Diseases and Diagnostic Laboratory, Minneapolis, MN, which serves three adjacent facilities (a children's hospital and low- and high-acuity adult facilities). ST131 accounted for 26% of the study isolates (more than any other clonal group), was distributed similarly by facility, and was closely associated with ciprofloxacin resistance and extended-spectrum ß-lactamase (ESBL) production. The H30 and H30Rx subclones accounted for most ST131 isolates and for the association of ST131 with fluoroquinolone resistance and ESBL production. Unlike ST131 per se, these subclones were distributed differentially by hospital, being most prevalent at the high-acuity adult facility and were absent from the children's hospital. The virulence gene profiles of ST131 and its subclones were distinctive and more extensive than those of other fluoroquinolone-resistant or ESBL-producing isolates. Within ST131, bla CTX-M-15 was confined to H30Rx isolates and other bla CTX-M variants to non-Rx H30 isolates. Pulsed-field gel electrophoresis documented a predominance of globally distributed pulsotypes and no local outbreak pattern. These findings help clarify the epidemiology, ecology, and bacterial correlates of the H30 and H30Rx ST131 subclones by documenting a high overall prevalence but significant segregation by facility, strong associations with fluoroquinolone resistance and specific ESBL variants, and distinctive virulence gene associations that may confer fitness advantages over other resistant E. coli.

Morphometric measurement of submucosal thickness in areas of fat deposition in the terminal ileum and colonic sections, with correlation with body mass index, weight and age: a male autopsy study.

AIMS: An increased amount of submucosal (SM) fat in the colon on imaging is considered to be characteristic of inflammatory bowel disease (IBD); however, a recent study in patients without IBD reported a correlation between colonic SM fat deposition and body weight (BW). The aim of this study was to perform a morphometric investigation of SM thickness in areas of fat deposition in the terminal ileum (TI), ileocaecal valve (ICV), and colonic sections, to determine whether there are variations by site, and whether it shows a correlation with BW, body mass index (BMI), or age. METHODS AND RESULTS: Representative samples of TI, ICV and colonic sections were collected prospectively from 115 autopsy cases without IBD. All of the study subjects were male (Veterans Hospital). SM thickness was measured in areas of fat deposition. Correlation analysis was performed between SM thickness and BW, BMI, and age. Fat deposition was common; however, with the exception of the ICV, it was neither consistent nor prominent, and it did not show a statistical correlation with BW, BMI, or age. CONCLUSIONS: SM fat deposition is common but not uniform or conspicuous in the TI or colon. In contrast to extravisceral intra-abdominal fat, it does not show a correlation with BW or BMI, and is not associated with ageing. As all study subjects were male, gender-dependent variability cannot be excluded.

Observational study of corrected count increments after transfusion of platelets treated with riboflavin pathogen reduction technology in additive solutions.

BACKGROUND: Mirasol pathogen reduction technology (PRT) treatment inactivates bacteria, viruses, and parasites in plasma products and platelets (PLTs) suspended in plasma and PLT additive solutions (PAS). Few clinical studies exist documenting transfusions with PAS. This study objective was to evaluate the count increments of PRT-treated PAS-C and PAS-E buffy coat (BC) PLTs in routine use observational settings. STUDY DESIGN AND METHODS: PLT pools of five or six BCs were collected, processed, and suspended in PAS-C or PAS-E, respectively. Products were exposed to ultraviolet light in the presence of riboflavin and then transfused into 19 patients with hematologic diseases. Patients were monitored for PLT corrected count increment (CCI) at 1 and 24 hours and for any adverse events in the 72 hours after transfusion. Sterility monitoring was performed with a microbial detection system (BacT/ALERT, bioMérieux). RESULTS: The PAS-E products had significantly higher PLT concentrations and counts than the PAS-C products. The mean CCIs of per-protocol (PP) units at 1 and 24 hours were 11,900 (n=27) and 5500 (n=30), respectively. Seventy-eight percent of PP transfusions classify as successful with CCIs at 1 hour of higher than 7500, and 63% higher than 4500 at 24 hours. One patient was excluded from all analyses as she was refractory to Mirasol-treated PLT transfusions and follow-up untreated transfusion products. No adverse events were observed and no contaminated products were detected by BacT/ALERT. CONCLUSION: PRT-treated BC PLTs in PAS-C or PAS-E demonstrate PLT transfusion success rates in hematology patients with thrombocytopenia that are comparable to previous studies examining PLTs stored in plasma.

Early diagnosis of intravascular large B-cell lymphoma: clues from routine blood smear morphologic findings.

Intravascular large B-cell lymphoma (IVLBCL) is a mature B-cell neoplasm characterized by malignant lymphoid cells within the lumina of blood vessels and capillaries. Given its varied and nonspecific clinical manifestation, this aggressive disease is often not diagnosed until an advanced clinical stage or even at autopsy. This case highlights a patient presenting with autoimmune hemolytic anemia (AIHA) and fevers. Atypical circulating cells on a screening peripheral smear lead to flow cytometric studies highlighting an increase in large, light chain restricted CD20 positive cells. A diagnostic bone marrow biopsy was performed and trephine cores demonstrated predominantly intrasinusoidal lymphoma cells. In conjunction with additional immunophenotypic data, these studies strongly supported a diagnosis of IVLBCL. Judicious use of flow cytometry and morphology resulted in an early-stage diagnosis and likely contributed to the patient's current complete remission status following anti-CD20 therapy. Differential diagnoses for this presentation are discussed in light of serologic, immunophenotypic, histologic, and cytogenetic findings.

New ß-lactamase inhibitors: a therapeutic renaissance in an MDR world.

As the incidence of Gram-negative bacterial infections for which few effective treatments remain increases, so does the contribution of drug-hydrolyzing ß-lactamase enzymes to this serious clinical problem. This review highlights recent advances in ß-lactamase inhibitors and focuses on agents with novel mechanisms of action against a wide range of enzymes. To this end, we review the ß-lactamase inhibitors currently in clinical trials, select agents still in preclinical development, and older therapeutic approaches that are being revisited. Particular emphasis is placed on the activity of compounds at the forefront of the developmental pipeline, including the diazabicyclooctane inhibitors (avibactam and MK-7655) and the boronate RPX7009. With its novel reversible mechanism, avibactam stands to be the first new ß-lactamase inhibitor brought into clinical use in the past 2 decades. Our discussion includes the importance of selecting the appropriate partner ß-lactam and dosing regimens for these promising agents. This "renaissance" of ß-lactamase inhibitors offers new hope in a world plagued by multidrug-resistant (MDR) Gram-negative bacteria.

Susceptibility to alternative oral antimicrobial agents in relation to sequence type ST131 status and Coresistance phenotype among recent Escherichia coli isolates from U.S. veterans.

The rising prevalence of resistance to first-line antimicrobial agents in Escherichia coli, which has paralleled the emergence of E. coli sequence type ST131, has created a need for alternative oral options for use in treating outpatients with infections such as cystitis and chronic prostatitis. Accordingly, we determined susceptibility to six alternative oral agents (azithromycin, chloramphenicol, doxycycline, fosfomycin, minocycline, and rifampin) by Etest or disk diffusion for 120 recently obtained E. coli clinical isolates from Veterans Affairs Medical Centers across the United States. Isolates were randomly selected in three subgroups of 40 isolates each based on coresistance to fluoroquinolones with and without extended-spectrum cephalosporins (ESCs). Results were stratified according to trimethoprim-sulfamethoxazole (TMP-SMZ) phenotype. Overall, the prevalence of susceptible (or susceptible plus intermediate) isolates varied by agent, with rifampin being lowest (0%), fosfomycin highest (98 to 99%), and others in the mid-range (37 to 88%). Substantial proportions of isolates (15 to 27%) yielded intermediate results for azithromycin, chloramphenicol, doxycycline, and minocycline. Among isolates resistant (versus susceptible) to fluoroquinolones with or without ESCs, susceptibility to the above four agents declined significantly among non-ST131 isolates but not ST131 isolates. In contrast, in the presence of resistance to TMP-SMZ, susceptibility to azithromycin, doxycycline, and minocycline was significantly reduced among both ST131 and non-ST131 isolates. These findings identify potential alternative oral agents for use with E. coli isolates resistant to fluoroquinolones, ESCs, and/or TMP-SMZ and suggest that determination of ST131 status could help guide initial antimicrobial selection, pending susceptibility results.

OxyR contributes to the virulence of a Clonal Group A Escherichia coli strain (O17:K+:H18) in animal models of urinary tract infection, subcutaneous infection, and systemic sepsis.

The oxidative stress response regulator OxyR was assessed as both a urinary and extra-urinary virulence factor in Escherichia coli strain UCB34 (O17:K+:H18), a representative of the emergent Clonal Group A (CGA). Compared to UCB34, the isogenic oxyR mutant exhibited increased H2O2 sensitivity, indistinguishable in vitro growth, and attenuated virulence in rodent models of urinary tract, subcutaneous infection, and systemic sepsis. Complemented mutants showed virulence levels comparable to parent strains in all models. These findings uniquely fulfill molecular Koch's postulates for a putative virulence factor of CGA, provide experimental evidence of an extra-urinary virulence promoting trait in CGA, and document a role for OxyR in local and systemic extra-urinary E. coli infections.

Novel ß-lactamase inhibitors: a therapeutic hope against the scourge of multidrug resistance.

The increasing incidence and prevalence of multi-drug resistance (MDR) among contemporary Gram-negative bacteria represents a significant threat to human health. Since their discovery, ß-lactam antibiotics have been a major component of the armamentarium against these serious pathogens. Unfortunately, a wide range of ß-lactamase enzymes have emerged that are capable of inactivating these powerful drugs. In the past 30 years, a major advancement in the battle against microbes has been the development of ß-lactamase inhibitors, which restore the efficacy of ß-lactam antibiotics (e.g., ampicillin/sulbactam, amoxicillin/clavulanate, ticarcillin/clavulanate, and piperacillin/tazobactam). Unfortunately, many newly discovered ß-lactamases are not inactivated by currently available inhibitors. Is there hope? For the first time in many years, we can anticipate the development and introduction into clinical practice of novel inhibitors. Although these inhibitors may still not be effective for all ß-lactamases, their introduction is still welcome. This review focuses on the novel ß-lactamase inhibitors that are closest to being introduced in the clinic.

Design and exploration of novel boronic acid inhibitors reveals important interactions with a clavulanic acid-resistant sulfhydryl-variable (SHV) ß-lactamase.

Inhibitor resistant (IR) class A ß-lactamases pose a significant threat to many current antibiotic combinations. The K234R substitution in the SHV ß-lactamase, from Klebsiella pneumoniae , results in resistance to ampicillin/clavulanate. After site-saturation mutagenesis of Lys-234 in SHV, microbiological and biochemical characterization of the resulting ß-lactamases revealed that only -Arg conferred resistance to ampicillin/clavulanate. X-ray crystallography revealed two conformations of Arg-234 and Ser-130 in SHV K234R. The movement of Ser-130 is the principal cause of the observed clavulanate resistance. A panel of boronic acid inhibitors was designed and tested against SHV-1 and SHV K234R. A chiral ampicillin analogue was discovered to have a 2.4 ± 0.2 nM K(i) for SHV K234R; the chiral ampicillin analogue formed a more complex hydrogen-bonding network in SHV K234R vs SHV-1. Consideration of the spatial position of Ser-130 and Lys-234 and this hydrogen-bonding network will be important in the design of novel antibiotics targeting IR ß-lactamases.

Crystal structure of a preacylation complex of the ß-lactamase inhibitor sulbactam bound to a sulfenamide bond-containing thiol-ß-lactamase.

The rise of inhibitor-resistant and other ß-lactamase variants is generating an interest in developing new ß-lactamase inhibitors to complement currently available antibiotics. To gain insight into the chemistry of inhibitor recognition, we determined the crystal structure of the inhibitor preacylation complex of sulbactam, a clinical ß-lactamase inhibitor, bound in the active site of the S70C variant of SHV-1 ß-lactamase, a resistance enzyme that is normally present in Klebsiella pneumoniae. The S70C mutation was designed to affect the reactivity of that catalytic residue to allow for capture of the preacylation complex. Unexpectedly, the 1.45 Å resolution inhibitor complex structure revealed that residue C70 is involved in a sulfenamide bond with K73. Such a covalent bond is not present in the wild-type SHV-1 or in an apo S70C structure also determined in this study. This bond likely contributed significantly to obtaining the preacylation complex with sulbactam due to further decreased reactivity toward substrates. The intact sulbactam is positioned in the active site such that its carboxyl moiety interacts with R244, S130, and T235 and its carbonyl moiety is situated in the oxyanion hole. To our knowledge, in addition to being the first preacylation inhibitor ß-lactamase complex, this is also the first observation of a sulfenamide bond between a cysteine and lysine in an active site. Not only could our results aid, therefore, structure-based inhibitor design efforts in class A ß-lactamases, but the sulfenamide-bond forming approach to yield preacylation complexes could also be applied to other classes of ß-lactamases and penicillin-binding proteins with the SXXK motif.

Inactivation of a class A and a class C ß-lactamase by 6ß-(hydroxymethyl)penicillanic acid sulfone.

ß-Lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) contribute significantly to the longevity of the ß-lactam antibiotics used to treat serious infections. In the quest to design more potent compounds and to understand the mechanism of action of known inhibitors, 6ß-(hydroxymethyl)penicillanic acid sulfone (6ß-HM-sulfone) was tested against isolates expressing the class A TEM-1 ß-lactamase and a clinically important variant of the AmpC cephalosporinase of Pseudomonas aeruginosa, PDC-3. The addition of the 6ß-HM-sulfone inhibitor to ampicillin was highly effective. 6ß-HM-sulfone inhibited TEM-1 with an IC(50) of 12 ± 2 nM and PDC-3 with an IC(50) of 180 ± 36 nM, and displayed lower partition ratios than commercial inhibitors, with partition ratios (k(cat)/k(inact)) equal to 174 for TEM-1 and 4 for PDC-3. Measured for 20 h, 6ß-HM-sulfone demonstrated rapid, first-order inactivation kinetics with the extent of inactivation being related to the concentration of inhibitor for both TEM-1 and PDC-3. Using mass spectrometry to gain insight into the intermediates of inactivation of this inhibitor, 6ß-HM-sulfone was found to form a major adduct of +247 ± 5 Da with TEM-1 and +245 ± 5 Da with PDC-3, suggesting that the covalently bound, hydrolytically stabilized acyl-enzyme has lost a molecule of water (HOH). Minor adducts of +88 ± 5 Da with TEM-1 and +85 ± 5 Da with PDC-3 revealed that fragmentation of the covalent adduct can result but appeared to occur slowly with both enzymes. 6ß-HM-sulfone is an effective and versatile ß-lactamase inhibitor of representative class A and C enzymes.

Resistance emergence mechanism and mechanism of resistance suppression by tobramycin for cefepime for Pseudomonas aeruginosa.

The panoply of resistance mechanisms in Pseudomonas aeruginosa makes resistance suppression difficult. Defining optimal regimens is critical. Cefepime is a cephalosporin whose 3' side chain provides some stability against AmpC ß-lactamases. We examined the activity of cefepime against P. aeruginosa wild-type strain PAO1 and its isogenic AmpC stably derepressed mutant in our hollow-fiber infection model. Dose-ranging studies demonstrated complete failure with resistance emergence (both isolates). Inoculum range studies demonstrated ultimate failure for all inocula. Lower inocula failed last (10 days to 2 weeks). Addition of a ß-lactamase inhibitor suppressed resistance even with the stably derepressed isolate. Tobramycin combination studies demonstrated resistance suppression in both the wild-type and the stably derepressed isolates. Quantitating the RNA message by quantitative PCR demonstrated that tobramycin decreased the message relative to that in cefepime-alone experiments. Western blotting with AmpC-specific antibody for P. aeruginosa demonstrated decreased expression. We concluded that suppression of ß-lactamase expression by tobramycin (a protein synthesis inhibitor) was at least part of the mechanism behind resistance suppression. Monte Carlo simulation demonstrated that a regimen of 2 g of cefepime every 8 h plus 7 mg/kg of body weight of tobramycin daily would provide robust resistance suppression for Pseudomonas isolates with cefepime MIC values up to 8 mg/liter and tobramycin MIC values up to 1 mg/liter. For P. aeruginosa resistance suppression, combination therapy is critical.

Exploring sequence requirements for C3/C4 carboxylate recognition in the Pseudomonas aeruginosa cephalosporinase: Insights into plasticity of the AmpC ß-lactamase.

In Pseudomonas aeruginosa, the chromosomally encoded class C cephalosporinase (AmpC ß-lactamase) is often responsible for high-level resistance to ß-lactam antibiotics. Despite years of study of these important ß-lactamases, knowledge regarding how amino acid sequence dictates function of the AmpC Pseudomonas-derived cephalosporinase (PDC) remains scarce. Insights into structure-function relationships are crucial to the design of both ß-lactams and high-affinity inhibitors. In order to understand how PDC recognizes the C3/C4 carboxylate of ß-lactams, we first examined a molecular model of a P. aeruginosa AmpC ß-lactamase, PDC-3, in complex with a boronate inhibitor that possesses a side chain that mimics the thiazolidine/dihydrothiazine ring and the C3/C4 carboxylate characteristic of ß-lactam substrates. We next tested the hypothesis generated by our model, i.e. that more than one amino acid residue is involved in recognition of the C3/C4 ß-lactam carboxylate, and engineered alanine variants at three putative carboxylate binding amino acids. Antimicrobial susceptibility testing showed that the PDC-3 ß-lactamase maintains a high level of activity despite the substitution of C3/C4 ß-lactam carboxylate recognition residues. Enzyme kinetics were determined for a panel of nine penicillin and cephalosporin analog boronates synthesized as active site probes of the PDC-3 enzyme and the Arg349Ala variant. Our examination of the PDC-3 active site revealed that more than one residue could serve to interact with the C3/C4 carboxylate of the ß-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme.

Modifications of the C6-substituent of penicillin sulfones with the goal of improving inhibitor recognition and efficacy.

In order to evaluate the importance of a hydrogen-bond donating substituent in the design of ß-lactamase inhibitors, a series of C6-substituted penicillin sulfones, lacking a C2' substituent, and having an sp(3) hybridized C6, was prepared and evaluated against a representative classes A and C ß-lactamases. It was found that a C6 hydrogen-bond donor is necessary for good inhibitory activity, but that this feature alone is not sufficient in this series of C6ß-substituted penicillin sulfones. Other factors which may impact the potency of the inhibitor include the steric bulk of the C6 substituent (e.g., methicillin sulfone) which may hinder recognition in the class A ß-lactamases, and also high similarity to the natural substrates (e.g., penicillin G sulfone) which may render the prospective inhibitor a good substrate of both classes of enzyme. The best inhibitors had non-directional hydrogen-bonding substituents, such as hydroxymethyl, which may allow sufficient conformational flexibility of the acyl-enzyme for abstraction of the C6 proton by E166 (class A), thus promoting isomerization to the ß-aminoacrylate as a stabilized acyl-enzyme.

Novel insights into the mode of inhibition of class A SHV-1 beta-lactamases revealed by boronic acid transition state inhibitors.

Boronic acid transition state inhibitors (BATSIs) are potent class A and C ß-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 ß-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 ß-lactamase with micromolar affinity that is considerably weaker than their inhibition of other ß-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other ß-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.

Design, synthesis, and crystal structures of 6-alkylidene-2'-substituted penicillanic acid sulfones as potent inhibitors of Acinetobacter baumannii OXA-24 carbapenemase.

Class D ß-lactamases represent a growing and diverse class of penicillin-inactivating enzymes that are usually resistant to commercial ß-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel ß-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2'-substituted penicillanic acid sulfones (1-5) were synthesized and tested against OXA-24, a clinically important ß-lactamase that inactivates carbapenems and is found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 ß-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC(50) values against OXA-24 and two OXA-24 ß-lactamase variants ranged from 10 ± 1 (4 vs WT) to 338 ± 20 nM (5 vs Tyr112Ala, Met223Ala). Compound 4 possessed the lowest K(i) (500 ± 80 nM vs WT), and 1 possessed the highest inactivation efficiency (k(inact)/K(i) = 0.21 ± 0.02 µM(-1) s(-1)). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 Å) reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanion hole. These data provide the first structural evidence that 6-alkylidene-2'-substituted penicillin sulfones are effective mechanism-based inactivators of class D ß-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D ß-lactamases is proposed.

Penicillin sulfone inhibitors of class D beta-lactamases.

OXA beta-lactamases are largely responsible for beta-lactam resistance in Acinetobacter spp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the beta-lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D beta-lactamases. To overcome this challenge, we explored the abilities of beta-lactamase inhibitors of the C-2- and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum (OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D beta-lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low K(i) values for the OXA-1 beta-lactamase (0.70 +/- 0.14 --> 1.60 +/- 0.30 microM) and demonstrated significant K(i) improvements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low K(i)s for the OXA-10, -14, -17, and -24/40 beta-lactamases (0.20 +/- 0.04 --> 17 +/- 4 microM). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.e., the partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation [t(n)] = 0) and t(n)s ranging from 5 to 8 for the other OXA enzymes. Using mass spectroscopy to study the intermediates in the inactivation pathway, we determined that JDB/LN-1-255 inhibited OXA beta-lactamases by forming covalent adducts that do not fragment. On the basis of the substrate and inhibitor kinetics of OXA-1, we constructed a model showing that the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213, the R-2 group at C-2 fits between the space created by the long B9 and B10 beta strands, and stabilizing hydrophobic interactions are formed between the pyridyl ring of JDB/LN-1-255 and Val116 and Leu161. By exploiting conserved structural and mechanistic features, JDB/LN-1-255 is a promising lead compound in the quest for effective inhibitors of OXA-type beta-lactamases.

Three decades of beta-lactamase inhibitors.

Since the introduction of penicillin, beta-lactam antibiotics have been the antimicrobial agents of choice. Unfortunately, the efficacy of these life-saving antibiotics is significantly threatened by bacterial beta-lactamases. beta-Lactamases are now responsible for resistance to penicillins, extended-spectrum cephalosporins, monobactams, and carbapenems. In order to overcome beta-lactamase-mediated resistance, beta-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) were introduced into clinical practice. These inhibitors greatly enhance the efficacy of their partner beta-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 beta-lactam-beta-lactamase inhibitor combinations. Furthermore, the prevalence of clinically relevant beta-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 beta-lactams. Here, we review the catalytic mechanisms of each beta-lactamase class. We then discuss approaches for circumventing beta-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 beta-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 beta-lactamases.