Acinetobacter-derived Cephalosporinase Research Articles

Proposal for Acinetobacter higginsii sp. nov. to accommodate organisms of human clinical origin previously classified as Acinetobacter genomic species 16.

In 1989, Bouvet and Jeanjean delineated five proteolytic genomic species (GS) of Acinetobacter, each with two to four human isolates. Three were later validly named, whereas the remaining two (GS15 and GS16) have been awaiting nomenclatural clarification. Here we present the results of the genus-wide taxonomic study of 13 human strains classified as GS16 (n=10) or GS15 (n=3). Based on core genome phylogenetic analysis, the strains formed two respective but closely related phylogroups within the Acinetobacter haemolytic clade. The intraspecies genomic average nucleotide identity based on blast (ANIb) values for GS16 and GS15 reached ≥94.9 % and ≥98.7, respectively, whereas ANIb values between them were 92.5-93.5% and those between them and the known species were ≤91.5 %. GS16 and GS15 could be differentiated from the other Acinetobacter species by their ability to lyse gelatin and sheep blood and to assimilate d,l-lactate, along with their inability to acidify d-glucose and assimilate glutarate. In contrast, GS16 and GS15 were indistinguishable from one another by metabolic/physiological features or whole-cell MALDI-TOF mass spectra. All the GS15/GS16 genomes contained genes encoding a class D β-lactamase, Acinetobacter-derived cephalosporinase and aminoglycoside 6'-N-acetyltransferase. Searching NCBI databases revealed genome sequences of three additional isolates of GS16, but none of GS15. We conclude that our data support GS16 as representing a novel species, but leave the question of the taxonomic status of GS15 open, given its close relatedness to GS16 and the small number of available strains. We propose the name Acinetobacter higginsii sp. nov. for GS16, with the type strain NIPH 1872T (CCM 9243T=CIP 70.18T=ATCC 17988T).

Synthesis of a Novel Boronic Acid Transition State Inhibitor, MB076: A Heterocyclic Triazole Effectively Inhibits Acinetobacter-Derived Cephalosporinase Variants with an Expanded-Substrate Spectrum.

Class C Acinetobacter-derived cephalosporinases (ADCs) represent an important target for inhibition in the multidrug-resistant pathogen Acinetobacter baumannii. Many ADC variants have emerged, and characterization of their structural and functional differences is essential. Equally as important is the development of compounds that inhibit all prevalent ADCs despite these differences. The boronic acid transition state inhibitor, MB076, a novel heterocyclic triazole with improved plasma stability, was synthesized and inhibits seven different ADC β-lactamase variants with Ki values <1 μM. MB076 acted synergistically in combination with multiple cephalosporins to restore susceptibility. ADC variants containing an alanine duplication in the Ω-loop, specifically ADC-33, exhibited increased activity for larger cephalosporins, such as ceftazidime, cefiderocol, and ceftolozane. X-ray crystal structures of ADC variants in this study provide a structural context for substrate profile differences and show that the inhibitor adopts a similar conformation in all ADC variants, despite small changes near their active sites.

Enhanced bacterial killing with colistin/sulbactam combination against carbapenem-resistant Acinetobacter baumannii

Aims: Polymyxin-based combination therapy is often used to treat carbapenem-resistant Acinetobacter baumannii (A. baumannii) infections. Although sulbactam is intrinsically active against A. baumannii, few studies have investigated colistin/sulbactam combinations against carbapenem-resistant A. baumannii.Methods: Whole genome sequencing was undertaken on eight carbapenem-resistant (colistin-susceptible) isolates of A. baumannii from Chinese patients. Bacterial killing of colistin and sulbactam, alone and in combination, was examined with checkerboard (all isolates) and static and dynamic time-kill studies (three isolates). In the dynamic studies, antibiotics were administered in various clinically-relevant dosing regimens that mimicked patient pharmacokinetics.Results: The eight isolates consisted of ST195, ST191 and ST208 belonging to clonal complex 208, which is the most epidemic clonal type of A. baumannii globally. All isolates possessed Acinetobacter-derived cephalosporinase (ADC-61 or ADC-78) and seven of eight isolates contained the carbapenem-resistance gene blaOXA-23. The colistin/sulbactam combination was synergistic against two of eight isolates in checkerboard studies. In time-kill studies, rapid bacterial killing of ca. 3–6 log10 CFU/mL was observed with colistin monotherapy, followed by steady regrowth. Sulbactam monotherapy was generally ineffective. Substantially enhanced bacterial killing was observed with colistin/sulbactam combinations in both static and dynamic models, especially with the higher sulbactam concentration (2 g) and/or longer sulbactam infusion time (2 hours) in the dynamic model.Conclusions: This study was the first to use a pharmacokinetics/pharmacodynamics model to investigate synergistic activity of colistin/sulbactam combinations against A. baumannii. It showed that clinically-relevant dosing regimens of colistin combined with sulbactam may substantially improve bacterial killing of multidrug-resistant and carbapenem-resistant A. baumannii.

Structural Insights into Inhibition of the Acinetobacter-Derived Cephalosporinase ADC-7 by Ceftazidime and Its Boronic Acid Transition State Analog.

Extended-spectrum class C β-lactamases have evolved to rapidly inactivate expanded-spectrum cephalosporins, a class of antibiotics designed to be resistant to hydrolysis by β-lactamase enzymes. To better understand the mechanism by which Acinetobacter-derived cephalosporinase-7 (ADC-7), a chromosomal AmpC enzyme, hydrolyzes these molecules, we determined the X-ray crystal structure of ADC-7 in an acyl-enzyme complex with the cephalosporin ceftazidime (2.40 Å) as well as in complex with a boronic acid transition state analog inhibitor that contains the R1 side chain of ceftazidime (1.67 Å). In the acyl-enzyme complex, the carbonyl oxygen is situated in the oxyanion hole where it makes key stabilizing interactions with the main chain nitrogens of Ser64 and Ser315. The boronic acid O1 hydroxyl group is similarly positioned in this area. Conserved residues Gln120 and Asn152 form hydrogen bonds with the amide group of the R1 side chain in both complexes. These complexes represent two steps in the hydrolysis of expanded-spectrum cephalosporins by ADC-7 and offer insight into the inhibition of ADC-7 by ceftazidime through displacement of the deacylating water molecule as well as blocking its trajectory to the acyl carbonyl carbon. In addition, the transition state analog inhibitor, LP06, was shown to bind with high affinity to ADC-7 (Ki , 50 nM) and was able to restore ceftazidime susceptibility, offering the potential for optimization efforts of this type of inhibitor.

1,2,3-Triazolylmethaneboronate: A Structure Activity Relationship Study of a Class of β-Lactamase Inhibitors against Acinetobacter baumannii Cephalosporinase.

Boronic acid transition state inhibitors (BATSIs) are known reversible covalent inhibitors of serine β-lactamases. The selectivity and high potency of specific BATSIs bearing an amide side chain mimicking the β-lactam’s amide side chain are an established and recognized synthetic strategy. Herein, we describe a new class of BATSIs where the amide group is replaced by a bioisostere triazole; these compounds were designed as molecular probes. To this end, a library of 26 α-triazolylmethaneboronic acids was synthesized and tested against the clinically concerning Acinetobacter-derived cephalosporinase, ADC-7. In steady state analyses, these compounds demonstrated Ki values ranging from 90 nM to 38 μM (±10%). Five compounds were crystallized in complex with ADC-7 β-lactamase, and all the crystal structures reveal the triazole is in the putative amide binding site, thus confirming the triazole–amide bioisosterism. The easy synthetic access of these new inhibitors as prototype scaffolds allows the insertion of a wide range of chemical groups able to explore the enzyme binding site and provides insights on the importance of specific residues in recognition and catalysis. The best inhibitor identified, compound 6q (Ki 90 nM), places a tolyl group near Arg340, making favorable cation−π interactions. Notably, the structure of 6q does not resemble the natural substrate of the β-lactamase yet displays a pronounced inhibition activity, in addition to lowering the minimum inhibitory concentration (MIC) of ceftazidime against three bacterial strains expressing class C β-lactamases. In summary, these observations validate the α-triazolylboronic acids as a promising template for further inhibitor design.

Distribution of carbapenem resistant Acinetobacter baumannii with blaADC-30 and induction of ADC-30 in response to beta-lactam antibiotics

A wide range of intrinsic Acinetobacter-derived cephalosporinases (ADC) along with other carbapenemases has now been detected in Acinetobacter baumannii leaving clinicians with few treatment options. The present study reports the spread of ADC-30 co-producing KPC-2 along with other β-lactamases among carbapenem resistant A. baumannii strains obtained from ICU patients in two Indian hospitals. Primer extension analysis revealed higher transcript level of the ADC gene when induced with cefoxitin at 8 μg/ml (170 fold), ceftriaxone at 8 μg/ml (136 fold), ceftazidime at 4 μg/ml (65 fold), cefepime at 8 μg/ml (77 fold) and aztreonam at 8 μg/ml (21 fold) when compared with the basal level without antibiotic pressure. Slight increase in expression of blaADC-30 when induced with imipenem and meropenem at 0.25 μg/ml (3 and 6 fold) was observed and may help in conferring resistance to carbapenem. MLST analysis revealed the circulation of A. baumannii sequence types ST188, ST386, ST583 and ST390 in these hospitals.

ISAba1 Regulated OXA-23 Carbapenem Resistance in Acinetobacter baumannii Strains in Durban, South Africa.

This study investigated the molecular mechanisms of resistance to carbapenems and cephalosporins in 24 consecutive, multidrug-resistant Acinetobacter baumannii (MDRAB) isolates collected between January and April 2015 by a private sector laboratory in Durban, South Africa. All isolates were resistant to all carbapenems tested. blaOXA-23 and blaOXA-51 genes were found in 23 isolates, while blaOXA-24, blaOXA-48, and blaOXA-58 were absent in all isolates. The most prevalent extended-spectrum β-lactamase was TEM-116 (92%). blaADC was present in 83.3% of isolates, of which two were new variants with three and five amino acid differences compared to Acinetobacter-derived cephalosporinase (ADC)-1, the first at positions 64E → K, 341N → T, and 342R → G and the second at positions 24G → D, 167S → P, 283R → F, 341N → T, and 342R → G, respectively. All isolates were negative for blaPER, blaCMY, blaGES, blaKPC, blaCTX-M, and blaSHV. Metallo-β-lactamase IMP and VIM were absent in all isolates, and NDM-1 was present in 1 isolate. ISAba1 was located upstream blaOXA-23 in all isolates and upstream blaADC (30, 78, 79, 87 and the ADC variants) in 54.2% of the ADC-carrying isolates. None of the isolates had ISAba1 inserted upstream blaOXA-51 gene. Four isolates were clonally related and showed two clusters (A and B), while 20 isolates remained unclustered. There was no direct relationship between the clusters and the hospitals they were isolated from. This study reports the first NDM-1-producing carbapenem resistant Acinetobacter baumannii isolate in South Africa and highlights the presence of OXA-23, the known ADCs (ADC-30, ADC-78, ADC-79, and ADC-87), and two new ADC variants associated with ISAba1 from the private health sector in Durban, South Africa. The complexity and diversity of MDRAB severely limit treatment options.

Structure-Based Analysis of Boronic Acids as Inhibitors of Acinetobacter-Derived Cephalosporinase-7, a Unique Class C β-Lactamase.

Acinetobacter baumannii is a multidrug resistant pathogen that infects more than 12 000 patients each year in the US. Much of the resistance to β-lactam antibiotics in Acinetobacter spp. is mediated by class C β-lactamases known as Acinetobacter-derived cephalosporinases (ADCs). ADCs are unaffected by clinically used β-lactam-based β-lactamase inhibitors. In this study, five boronic acid transition state analog inhibitors (BATSIs) were evaluated for inhibition of the class C cephalosporinase ADC-7. Our goal was to explore the properties of BATSIs designed to probe the R1 binding site. Ki values ranged from low micromolar to subnanomolar, and circular dichroism (CD) demonstrated that each inhibitor stabilizes the β-lactamase-inhibitor complexes. Additionally, X-ray crystal structures of ADC-7 in complex with five inhibitors were determined (resolutions from 1.80 to 2.09 Å). In the ADC-7/CR192 complex, the BATSI with the lowest Ki (0.45 nM) and greatest Δ Tm (+9 °C), a trifluoromethyl substituent, interacts with Arg340. Arg340 is unique to ADCs and may play an important role in the inhibition of ADC-7. The ADC-7/BATSI complexes determined in this study shed light into the unique recognition sites in ADC enzymes and also offer insight into further structure-based optimization of these inhibitors.

Inhibition of Acinetobacter-Derived Cephalosporinase: Exploring the Carboxylate Recognition Site Using Novel β-Lactamase Inhibitors.

Boronic acids are attracting a lot of attention as β-lactamase inhibitors, and in particular, compound S02030 ( Ki = 44 nM) proved to be a good lead compound against ADC-7 ( Acinetobacter-derived cephalosporinase), one of the most significant resistance determinants in A.baumannii. The atomic structure of the ADC-7/S02030 complex highlighted the importance of critical structural determinants for recognition of the boronic acids. Herein, to elucidate the role in recognition of the R2-carboxylate, which mimics the C3/C4 found in β-lactams, we designed, synthesized, and characterized six derivatives of S02030 (3a). Out of the six compounds, the best inhibitors proved to be those with an explicit negative charge (compounds 3a-c, 3h, and 3j, Ki = 44-115 nM), which is in contrast to the derivatives where the negative charge is omitted, such as the amide derivative 3d ( Ki = 224 nM) and the hydroxyamide derivative 3e ( Ki = 155 nM). To develop a structural characterization of inhibitor binding in the active site, the X-ray crystal structures of ADC-7 in a complex with compounds 3c, SM23, and EC04 were determined. All three compounds share the same structural features as in S02030 but only differ in the carboxy-R2 side chain, thereby providing the opportunity of exploring the distinct binding mode of the negatively charged R2 side chain. This cephalosporinase demonstrates a high degree of versatility in recognition, employing different residues to directly interact with the carboxylate, thus suggesting the existence of a "carboxylate binding region" rather than a binding site in ADC enzymes. Furthermore, this class of compounds was tested against resistant clinical strains of A.baumannii and are effective at inhibiting bacterial growth in conjunction with a β-lactam antibiotic.

Antibiotic resistance of pathogenic Acinetobacter species and emerging combination therapy.

The increasing antibiotic resistance of Acinetobacter species in both natural and hospital environments has become a serious problem worldwide in recent decades. Because of both intrinsic and acquired antimicrobial resistance (AMR) against last-resort antibiotics such as carbapenems, novel therapeutics are urgently required to treat Acinetobacter-associated infectious diseases. Among the many pathogenic Acinetobacter species, A. baumannii has been reported to be resistant to all classes of antibiotics and contains many AMR genes, such as bla ADC (Acinetobacter-derived cephalosporinase). The AMR of pathogenic Acinetobacter species is the result of several different mechanisms, including active efflux pumps, mutations in antibiotic targets, antibiotic modification, and low antibiotic membrane permeability. To overcome the limitations of existing drugs, combination theraphy that can increase the activity of antibiotics should be considered in the treatment of Acinetobacter infections. Understanding the molecular mechanisms behind Acinetobacter AMR resistance will provide vital information for drug development and therapeutic strategies using combination treatment. Here, we summarize the classic mechanisms of Acinetobacter AMR, along with newly-discovered genetic AMR factors and currently available antimicrobial adjuvants that can enhance drug efficacy in the treatment of A. baumannii infections.

The Novel β-Lactamase Inhibitor, ETX-2514, in Combination with Sulbactam Effectively Inhibits Acinetobacter baumannii

BackgroundMultidrug resistant (MDR) Acinetobacter sp. were deemed a “serious” health threat by the Centers for Disease Control and Prevention with a daunting 63% of infections being nearly untreatable. Underlying this challenging pathogen are the presence of a chromosomal class C β-lactamase, Acinetobacter-derived cephalosporinase (ADC), as well as the abundant prevalence of class D OXA β-lactamases that hydrolyze carbapenems in conjunction with a lack of potent β-lactamase inhibitors. Based on the ability of ETX2514, a rationally designed novel diazabicyclooctane inhibitor (Figure 1A), to inhibit class D β-lactamases as well as PBPs, we hypothesized that highly resistant clinical isolates of A. baumannii will demonstrate susceptibility to the sulbactam-ETX2514 combination and that A. baumannii β-lactamases, ADC-7 and OXA-58 will be readily inactivated by ETX2514.MethodsSusceptibility testing according to Clinical and Laboratory Standards Institute was performed for sulbactam ±4 mg/L ETX2514 using 72 A. baumannii strains. More than half of the isolates are MDR, have ≥ eight resistant determinants, contain an ADC β-lactamase, and have OXA-23 and OXA-58-like β-lactamases in the carbapenem resistant isolates. ADC-7 and OXA-58 β-lactamases were purified and characterized with ETX2514 by steady-state inhibition kinetics and Q-TOF mass spectrometry.ResultsThe A. baumannii strains demonstrated an MIC90 of 32 mg/L for sulbactam and 2 mg/L for the sulbactam-ETX-2514 combination. The addition of ETX2514 lowered the MIC50 from 8 to 1 mg/L (Figure 1B). ETX-2514 effectively inhibited purified OXA-58 (k2/K = 2.5 ± 0.3 × 105 M−1 s−1 and Ki apP = 0.39 ± 0.01 µM) and ADC-7 (k2/K =1.0 ± 0.1 × 106 M−1 s−1 and Ki app = 0.11 ± 0.04 µM). The two β-lactamases displayed similar dissociation constants (Kd =1 ± 0.1 nM), but ADC-7 possessed a faster dissociation rate (koff =8 ± 1 × 10−4 s−1 for ADC-7 and 1.6 ± 0.3 × 10−4 s−1for OXA-58). Using mass spectrometry, ADC-7 and OXA-58 were found to stably bind the ETX-2514 compound for up to 24 hours (Figure 1C).ConclusionETX2514 is a new β-lactamase inhibitor that is strikingly effective at restoring susceptibility to highly drug-resistant A. baumannii isolates when combined with sulbactam via inhibition of the ADC-7 and OXA-58 β-lactamases.Disclosures F. Van Den Akker, Entasis: Grant Investigator, Research grant Wockhardt: Grant Investigator, Research grant; K. M. Papp-Wallace, Entasis: Grant Investigator, Research grant Allecra: Grant Investigator, Research grant Merck: Grant Investigator, Research grant Roche: Grant Investigator, Research grant Allergan: Grant Investigator, Research grant; R. A. Bonomo, Entasis: Grant Investigator, Research grant Allecra: Grant Investigator, Research grant Wockhardt: Grant Investigator, Research grant Merck: Grant Investigator, Research grant Roche: Grant Investigator, Research grant GSK: Grant Investigator, Research grant Allergan: Grant Investigator, Research grant Shionogi: Grant Investigator, Research grant

Detection of AmpC β-lactamases in Acinetobacter baumannii in the Xuzhou region and analysis of drug resistance.

The aim of the present study was to determine the prevalence and related drug resistance of AmpC β-lactamases in Acinetobacter baumannii in tertiary-level hospitals in the Xuzhou region in China. A total of 134 clinical isolates of non-repetitive Acinetobacter baumannii were collected from different hospitals in the Xuzhou region, and multiplex polymerase chain reaction (PCR) was employed to determine the genotype of AmpC. The PCR products were purified and sequenced. The susceptibility to antibiotics was tested using the biometrics automated microbiological-assay system, VITEK-2. Amongst the 134 isolated strains, 96 strains were found to produce AmpC β-lactamases, and the positive rate was 72%, all of which carried acinetobacter-derived cephalosporinase (ADC) type AmpC resistance genes. The drug sensitivity tests indicated that the positive Acinetobacter baumannii strains were resistant to the majority of extended-spectrum β-lactam antibiotics, but were only sensitive to polymyxin. In conclusion, the incidence of AmpC enzymes in Acinetobacter baumannii strains in tertiary-level hospitals in the Xuzhou area is relatively high, and resistance to the majority of extended-spectrum β-lactam antibiotics may be related to the ADC type of AmpC.

Spread of imipenem-resistant Acinetobacter baumannii co-expressing OXA-23 and GES-11 carbapenemases in Lebanon

The acquisition of carbapenemases by Acinetobacter baumannii is reported increasingly worldwide, but data from Lebanon are limited. The aims of this study were to evaluate the prevalence of imipenem-resistant A. baumannii in Lebanon, identify resistance determinants, and detect clonal relatedness. Imipenem-resistant A. baumannii were collected from nine Lebanese hospitals during 2012. Antimicrobial susceptibility, the cloxacillin effect, and ethylenediaminetetraacetic acid (EDTA) synergy were determined. Genes encoding carbapenemases and insertion sequence ISAba1 were screened via PCR sequencing. ISAba1 position relative to genes encoding Acinetobacter-derived cephalosporinases (ADCs) and OXA-23 was studied by PCR mapping. Clonal linkage was examined by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Out of 724 A. baumannii isolated in 2012, 638 (88%) were imipenem-resistant. Of these, 142 were analyzed. Clavulanic acid-imipenem synergy suggested carbapenem-hydrolyzing extended-spectrum β-lactamase. A positive cloxacillin test indicated ADCs, while EDTA detection strips were negative. Genotyping indicated that 90% of isolates co-harbored blaOXA-23 and blaGES-11. The remaining strains had blaOXA-23, blaOXA-24, blaGES-11, or blaOXA-24 with blaGES-11. ISAba1 was located upstream of blaADC and blaOXA-23 in 97% and 100% of isolates, respectively. ERIC-PCR fingerprinting revealed 18 pulsotypes spread via horizontal gene transfer and clonal dissemination. This survey established baseline evidence of OXA-23 and GES-11-producing A. baumannii in Lebanon, indicating the need for further surveillance.

Contribution of Acinetobacter-derived cephalosporinase-30 to sulbactam resistance in Acinetobacter baumannii.

The sulbactam resistance rate in Acinetobacter baumannii has increased worldwide. Previous reports have shown that the β-lactamase blaTEM-1 confers resistance to sulbactam in A. baumannii. The purpose of this study was to examine whether other β-lactamases, including the Acinetobacter-derived cephalosporinase (ADC), OXA-23, OXA-24/72, and OXA-58 families, also contribute to sulbactam resistance in A. baumannii. The correlation between these β-lactamases and the sulbactam minimal inhibitory concentration (MIC) was determined using A. baumannii clinical isolates from diverse clonality, which were collected in a nationwide surveillance program from 2002 to 2010 in Taiwan. A possible association between the genetic structure of ISAba1-blaADC-30 and sulbactam resistance was observed because this genetic structure was detected in 97% of sulbactam-resistant strains compared with 10% of sulbactam-susceptible strains. Transformation of ISAba1-blaADC-30 into susceptible strains increased the sulbactam MIC from 2 to 32 μg/ml, which required blaADC-30 overexpression using an upstream promoter in ISAba1. Flow cytometry showed that ADC-30 production increased in response to sulbactam, ticarcillin, and ceftazidime treatment. This effect was regulated at the RNA level but not by an increase in the blaADC-30 gene copy number as indicated by quantitative PCR. Purified ADC-30 decreased the inhibitory zone created by sulbactam or ceftazidime, similarly to TEM-1. In conclusion, ADC-30 overexpression conferred resistance to sulbactam in diverse clinical A. baumannii isolates.

Biochemical and structural analysis of inhibitors targeting the ADC-7 cephalosporinase of Acinetobacter baumannii.

β-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C β-lactamase enzymes, known as Acinetobacter-derived cephalosporinases (ADCs). Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C cephalosporinase. Herein, two boronic acid transition state inhibitors (BATSIs S02030 and SM23) that are chemically distinct from β-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal cephalosporinase from Acinetobacter baumannii (Ki = 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 Å resolution) and in complex with S02030 (2.0 Å resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030 is bound in the oxyanion hole, and the R1 amide group makes key interactions with conserved residues Asn152 and Gln120. In addition, the carboxylate group of the inhibitor is meant to mimic the C3/C4 carboxylate found in β-lactams. The C3/C4 carboxylate recognition site in class C enzymes is comprised of Asn346 and Arg349 (AmpC numbering), and these residues are conserved in ADC-7. Interestingly, in the ADC-7/S02030 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme AmpC. A thermodynamic analysis suggests that ΔH driven compounds may be optimized to generate new lead agents. The ADC-7/BATSI complex provides insight into recognition of non-β-lactam inhibitors by ADC enzymes and offers a starting point for the structure-based optimization of this class of novel β-lactamase inhibitors against a key resistance target.

The X‐ray crystal structure of the Acinetobacter‐Derived Cephalosporinase, ADC‐7, at 1.7 Å

β‐Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. The Acinetobacter‐Derived Cephalosporinases (ADCs) are class C β‐lactamases found in A. baumannii and other Acinetobacter species that are responsible for resistance to penicillins, cephalosporins, and β‐lactam‐β‐lactamase inhibitor combinations. In order to probe the mechanism of substrate turnover, as well as to design novel β‐lactam antibiotics, it was important to elucidate the protein structure of an ADC enzyme. Here, we report the successful purification, crystallization, and determination of the crystal structure of ADC‐7 at 1.7 Å. This complete structure allows for the critical comparison of the overall structure and active site architecture of ADC‐7 with the known cephalosporinase, AmpC. Hopefully, our work will contribute to the development of a structure/function relationship for ADC‐7 that will provide insight into bacterial antibiotic resistance.

Multi-Drug Resistant Acinetobacter-Derived Cephalosporinase and OXAsetC Genes in Clinical Specimens of Acinetobacter spp. Isolated From Teaching Hospital

Background: Hospital-acquired infections caused by multi-drug resistant Acinetobacter spp. are often extremely difficult to treatand this has proved to be a serious problem worldwide. Objectives: The aim of this study was to determine the incidence rates and distribution patterns of multi-drug resistant (MDR) Acinetobacter spp. strains and the occurrence of Acinetobacter-derived cephalosporinase (ADC-7) and OXA-type carbapenemases (OXAsetC genes) in clinical specimens in the Beheshti Teaching Hospital in Kashan, Iran. Materials and Methods: This descriptive study was carried out on sixty isolates of Acinetobacter spp. and clinical samples collected from patients. The level of antibiotic resistance was determined by the disc diffusion method and the results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) procedure. Polymerase chain reaction (PCR) amplification of the genetic determinants of resistance was also determined. Results: The resistance rates were; amikacin (80%), tobramycin (68.3%), ceftazidime (60%), ciprofloxacin (55%), piperacillin/tazobactam (51.7%), doxycycline (50%), SXT/TMP (sulfamethoxazole / trimethoprim)(48.3%), levofloxacin (43.3%), gentamicin (40%), imipenem (25%), and sulbactam/ampicillin (20%). The frequency of MDR Acinetobacter spp. strains isolated was found to be 56.7%. These isolates were most sensitive to imipenem followed by ampicillin/sulbactam and gentamicin. The prevalence of genes for ADC-7 and OXAsetC in the Acinetobacter spp. was; 34 (56.7%) and 32 (53.3%), respectively. The positive percentages of MDR isolates for ADC-7 were 82.4% and for OXAsetC they were 73.5%. Conclusions: Our phenotypic analysis demonstrated that Acinetobacter spp. isolates were resistant to most clinically significant antibiotic classes. This is the first report concerning Acinetobacter-derived cephalosporinase, blaADC, enzymes in Acinetobacter spp. isolates from Iran.

Epidemiology of the Acinetobacter-derived cephalosporinase, carbapenem-hydrolysing oxacillinase and metallo-β-lactamase genes, and of common insertion sequences, in epidemic clones of Acinetobacter baumannii from Spain

To study the distribution, diversity and activity of Acinetobacter-derived cephalosporinase (ADC)-, carbapenem-hydrolysing oxacillinase (CHO)- and metallo-β-lactamase (MBL)-encoding genes, and of the most common insertion sequences (ISs), in the genome of nosocomial, epidemic, multidrug-resistant Acinetobacter baumannii (MDRAB) clones from Spain. The studied population included 59 MDRAB strains previously genotyped by PFGE and multilocus sequence typing. The search for the ADC (bla(ADC)), CHO (bla(OXA-51-like), bla(OXA-23-like), bla(OXA-40-like) and bla(OXA-58-like)) and MBL (bla(IMP), bla(VIM), bla(SIM-1), bla(GIM-1), bla(SPM-1) and bla(NDM-1)) genes, and for the ISs (ISAba1, ISAba2, ISAba3, ISAba4 and IS18) was done by PCR assays. The phenotypic presence of MBL enzymes was examined using imipenem/imipenem + EDTA strips. The most prevalent IS, ISAba1 (93.2%), was detected upstream of bla(ADC) and bla(OXA-51-like). These genes showed ample diversity (10 and 8 alleles, respectively). Four ADC sequences (ADC-1-like(P240S), ADC-2-like(N260H/T264N), ADC-11-like(Q163K) and ADC-11-like(G342R)) are described here for the first time. bla(OXA-58-like) was carried by 20.3% of strains, in association with ISAba2, ISAba3 or IS18. bla(OXA-40-like) was the most prevalent acquired CHO gene (57.6%), and was associated with none of the studied ISs. Neither bla(OXA-23-like) nor ISAba4 was detected in any strain. Some 67.8% of strains with MBL activity showed no corresponding gene in PCR; these results were more common in strains with a highly active CHO, such as OXA-40. All the studied genes and their related ISs showed a clonal distribution. Imipenem resistance was probably provided by OXA-40 for the most part, while MBL- and OXA-23-encoding genes were absent in the studied population.

Dissemination of ceftazidime-resistant Acinetobacter baumannii clonal complex 92 in Korea

This study was performed to describe the epidemiological traits of ceftazidime-resistant Acinetobacter baumannii clinical isolates from Korea. Antimicrobial susceptibilities were determined by disk diffusion assay. PCR experiments were performed to detect genes encoding extended-spectrum β-lactamases and metallo-β-lactamases. Detection of ISAba1 upstream of the bla(ADC) gene was also performed by PCR amplification. The genetic organization of the bla(PER-1) gene was investigated by PCR mapping and sequencing of the regions surrounding the gene. Multilocus sequence typing was performed using seven housekeeping genes. A. baumannii isolates of clonal complex (CC) 92 exhibited a higher resistance rate (286/289, 99%) against ceftazidime compared to A. baumannii isolates of non-CC92 (7/87, 8%). Amongst 286 ceftazidime-resistant isolates of CC92, 100 (35%) isolates carried the bla(PER-1) gene, while none of the 87 isolates of non-CC92 carried the gene. The bla(ADC) gene associated with an ISAba1 element was detected in 98% (281/286) of ceftazidime-resistant isolates of CC92 and in all seven ceftazidime-resistant isolates of non-CC92. The bla(PER-1) gene was located on a transposon, Tn1213 (ISPa12-bla(PER-1) -Δgst-ISPa13), in 95 isolates and on a complex class 1 integron (orf513-bla(PER-1) -putative ABC transporter gene) in five isolates. Southern blot experiments confirmed the chromosomal location of the bla(PER-1) gene. Acinetobacter baumannii CC92 which has acquired ceftazidime resistance by the production of PER-1 extended-spectrum β-lactamases and/or the overproduction of Acinetobacter-derived cephalosporinase is widely disseminated in Korea. This study shows the mechanisms of acquiring ceftazidime resistance in A. baumannii and the epidemiological traits of ceftazidime-resistant A. baumannii isolates from Korea.

Inhibition of the Class C β-Lactamase from Acinetobacter spp.: Insights into Effective Inhibitor Design

The need to develop beta-lactamase inhibitors against class C cephalosporinases of Gram-negative pathogens represents an urgent clinical priority. To respond to this challenge, five boronic acid derivatives, including a new cefoperazone analogue, were synthesized and tested against the class C cephalosporinase of Acinetobacter baumannii [Acinetobacter-derived cephalosporinase (ADC)]. The commercially available carbapenem antibiotics were also assayed. In the boronic acid series, a chiral cephalothin analogue with a meta-carboxyphenyl moiety corresponding to the C(3)/C(4) carboxylate of beta-lactams showed the lowest K(i) (11 +/- 1 nM). In antimicrobial susceptibility tests, this cephalothin analogue lowered the ceftazidime and cefotaxime minimum inhibitory concentrations (MICs) of Escherichia coli DH10B cells carrying bla(ADC) from 16 to 4 microg/mL and from 8 to 1 microg/mL, respectively. On the other hand, each carbapenem exhibited a K(i) of <20 microM, and timed electrospray ionization mass spectrometry (ESI-MS) demonstrated the formation of adducts corresponding to acyl-enzyme intermediates with both intact carbapenem and carbapenem lacking the C(6) hydroxyethyl group. To improve our understanding of the interactions between the beta-lactamase and the inhibitors, we constructed models of ADC as an acyl-enzyme intermediate with (i) the meta-carboxyphenyl cephalothin analogue and (ii) the carbapenems, imipenem and meropenem. Our first model suggests that this chiral cephalothin analogue adopts a novel conformation in the beta-lactamase active site. Further, the addition of the substituent mimicking the cephalosporin dihydrothiazine ring may significantly improve affinity for the ADC beta-lactamase. In contrast, the ADC-carbapenem models offer a novel role for the R(2) side group and also suggest that elimination of the C(6) hydroxyethyl group by retroaldolic reaction leads to a significant conformational change in the acyl-enzyme intermediate. Lessons from the diverse mechanisms and structures of the boronic acid derivatives and carbapenems provide insights for the development of new beta-lactamase inhibitors against these critical drug resistance targets.