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BackgroundPseudomonas aeruginosa may develop resistance to novel cephalosporin/β-lactamase inhibitor combinations during therapy through the acquisition of structural mutations in AmpC.ObjectivesTo describe the molecular and biochemical mechanisms involved in the development of resistance to ceftolozane/tazobactam in vivo through the selection and overproduction of a novel AmpC variant, designated PDC-315.MethodsPaired susceptible/resistant isolates obtained before and during ceftolozane/tazobactam treatment were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. Characterization of the novel PDC-315 variant was performed through genotypic and biochemical studies. The effects at the molecular level of the Asp245Asn change were analysed by molecular dynamics simulations using Amber.ResultsWGS identified mutations leading to modification (Asp245Asn) and overproduction of AmpC. Susceptibility testing revealed that PAOΔC producing PDC-315 displayed increased MICs of ceftolozane/tazobactam, decreased MICs of piperacillin/tazobactam and imipenem and similar susceptibility to ceftazidime/avibactam compared with WT PDCs. The catalytic efficiency of PDC-315 for ceftolozane was 10-fold higher in relation to the WT PDCs, but 3.5- and 5-fold lower for piperacillin and imipenem. IC50 values indicated strong inhibition of PDC-315 by avibactam, but resistance to cloxacillin inhibition. Analysis at the atomic level explained that the particular behaviour of PDC-315 is linked to conformational changes in the H10 helix that favour the approximation of key catalytic residues to the active site.ConclusionsWe deciphered the precise mechanisms that led to the in vivo emergence of resistance to ceftolozane/tazobactam in P. aeruginosa through the selection of the novel PDC-315 enzyme. The characterization of this new variant expands our knowledge about AmpC-mediated resistance to cephalosporin/β-lactamase inhibitors in P. aeruginosa.

The global distribution of carbapenemases such as KPC, OXA-48, and metallo-β-lactamases (MBLs) gives cause for concern, as these enzymes are not inhibited by classical β-lactamase inhibitors (BLIs). The current development of new inhibitors is one of the most promising highlights for the treatment of multidrug-resistant bacteria. The activity of cefepime in combination with the novel BLIs zidebactam, taniborbactam, and enmetazobactam was studied in a collection of 400 carbapenemase-producing Enterobacterales (CPE). The genomes were fully sequenced and potential mechanisms of resistance to cefepime/BLI combinations were characterized. Cefepime resistance in the whole set of isolates was 79.5% (MIC50/90 64/≥128mg/L). The cefepime/zidebactam and cefepime/taniborbactam combinations showed the highest activity (MIC(50/90) ≤0.5/1 and ≤0.5/2 mg/L, respectively). Cefepime/zidebactam displayed high activity, regardless of the carbapenemase or extended-spectrum β-lactamase (ESBL) considered (99% of isolates displayed MIC ≤2 mg/L). Cefepime/taniborbactam displayed excellent activity against OXA-48- and KPC-producing Enterobacterales and lower activity against MBL-producing isolates (four strains yielded MICs ≥16 mg/L: 2 NDM producers with an insertion in PBP3, one VIM-1 producer with nonfunctional OmpK35, and one IMP-8 producer). Cefepime/enmetazobactam displayed the lowest activity (MIC(50/90) 1/≥128 mg/L), with MICs ≥16 mg/L for 49 MBL producers, 40 OXA-48 producers (13 with amino acid changes in OmpK35/36, 4 in PBPs and 11 in RamR) and 25 KPC producers (most with an insertion in OmpK36). These results confirm the therapeutic potential of the new β-lactamase inhibitors, shedding light on the activity of cefepime and BLIs against CPE and resistance mechanisms. The cefepime/zidebactam and cefepime/taniborbactam combinations are particularly highlighted as promising alternatives to penicillin-based inhibitors for the treatment of CPE.

BackgroundLpxB is an enzyme involved in the biosynthesis pathway of lipid A, a component of LPS.ObjectivesTo evaluate the lpxB gene in Acinetobacter baumannii as a potential therapeutic target and to propose antisense agents such as peptide nucleic acids (PNAs) as a tool to combat bacterial infection, either alone or in combination with known antimicrobial therapies.MethodsRNA-seq analysis of the A. baumannii ATCC 17978 strain in a murine pneumonia model was performed to study the in vivo expression of lpxB. Protein expression was studied in the presence or absence of anti-lpxB (KFF)3K-PNA (pPNA). Time–kill curve analyses and protection assays of infected A549 cells were performed. The chequerboard technique was used to test for synergy between pPNA and colistin. A Galleria mellonella infection model was used to test the in vivo efficacy of pPNA.ResultsThe lpxB gene was overexpressed during pneumonia. Treatment with a specific pPNA inhibited LpxB expression in vitro, decreased survival of the ATCC 17978 strain and increased the survival rate of infected A549 cells. Synergy was observed between pPNA and colistin in colistin-susceptible strains. In vivo assays confirmed that a combination treatment of anti-lpxB pPNA and colistin was more effective than colistin in monotherapy.ConclusionsThe lpxB gene is essential for A. baumannii survival. Anti-lpxB pPNA inhibits LpxB expression, causing bacterial death. This pPNA showed synergy with colistin and increased the survival rate in G. mellonella. The data suggest that antisense pPNA molecules blocking the lpxB gene could be used as antibacterial agents.

Treatment of infections caused by Acinetobacter spp., particularly A. baumannii, is a major clinical problem due to its high rates of antibiotic resistance. New strategies must be developed, therefore, restoration of β-lactam efficacy through the use of β-lactamase inhibitors is paramount. Activities of the antibiotics imipenem, meropenem, cefepime, and sulbactam in combination with the penicillin-sulfone inhibitor LN-1-255 were tested by microdilution against 148 isolates of Acinetobacter spp. collected in 14 hospitals in Spain in 2020. Relevantly, the MIC90 (i.e., minimum concentration at which 90% of isolates were inhibited) of antibiotics in combination with LN-1-255 decreased 4- to 8-fold for all of the Acinetobacter isolates. Considering only the carbapenem-resistant A. baumannii isolates, which produce carbapenem-hydrolyzing class D β-lactamases, the addition of LN-1-255 decreased the resistance rates from 95.1% to 0% for imipenem, from 100% to 9.8% for meropenem, from 70.7% to 7.3% for cefepime, and sulbactam resistance rates from 9.8% to 0% and intermediate susceptibility rates from 53.7% to 2.4%. The inhibitor also decreased the minimum inhibitory concentrations (MICs) when tested against non-carbapenem-resistant Acinetobacter spp. isolates. In conclusion, combining LN-1-255 with imipenem, meropenem, cefepime, and sulbactam to target A. baumannii, and especially carbapenem-resistant isolates, represents an attractive option that should be developed for the treatment of infections caused by this pathogen.

Carbapenem resistance is a major global health problem that seriously compromises the treatment of infections caused by nosocomial pathogens. Resistance to carbapenems mainly occurs via the production of carbapenemases, such as VIM, IMP, NDM, KPC and OXA, among others. Preclinical and clinical trials are currently underway to test a new generation of promising inhibitors, together with the recently approved avibactam, relebactam and vaborbactam. This review summarizes the main, most promising carbapenemase inhibitors synthesized to date, as well as their spectrum of activity and current stage of development. We particularly focus on β-lactam/β-lactamase inhibitor combinations that could potentially be used to treat infections caused by carbapenemase-producer pathogens of critical priority. The emergence of these new combinations represents a step forward in the fight against antimicrobial resistance, especially in regard to metallo-β-lactamases and carbapenem-hydrolysing class D β-lactamases, not currently inhibited by any clinically approved inhibitor.

The emergence of 16S rRNA methyltransferases (RMTs) in gram-negative pathogens bearing other clinically relevant resistance mechanisms, such as carbapenemase-producing Enterobacterales (CPE), is becoming an alarming concern. We investigated the prevalence, antimicrobial susceptibility, resistance mechanisms, molecular epidemiology, and genetic support of RMTs in CPE isolates from Spain. The study included a collection of 468 CPE isolates recovered during 2018 from 32 participating Spanish hospitals. MICs were determined using the broth microdilution method, the agar dilution method (fosfomycin) or MIC gradient strips (plazomicin). All isolates were subjected to hybrid whole-genome sequencing. Sequence types (STs), core-genome phylogenetic relatedness, horizontally acquired resistance mechanisms, plasmid analysis and genetic environment of RMTs was in silico determined from WGS data in all RMT-positive isolates. Among the 468 CPE isolates evaluated, 24 (5.13%) isolates recovered from 9 different hospitals spanning 5 different Spanish regions showed resistance to all aminoglycosides and were positive for an RMT: 21 RmtF, 2 AmrA and 1 RmtC. All the RMT-producers showed high-level resistance to all aminoglycosides, including plazomicin, and in most of cases exhibited an extensively drug-resistant (XDR) susceptibility profile. The RMT-positive isolates showed low genetic diversity and were global clones of K. pneumoniae (ST147, ST101, ST395) or E. cloacae (ST93) species bearing blaOXA-48, blaNDM-1 or blaVIM-1 carbapenemase genes. RMTs were in 5 different multidrug-resistant plasmids and linked to efficient mobile elements. Our findings highlight that RMTs are emerging among clinical CPE isolates from Spain and their spread should be monitored to preserve the clinical utility of aminoglycosides and plazomicin in the future.

Background Imipenem/relebactam is a novel carbapenem/β-lactamase inhibitor combination, developed to act against carbapenemase-producing Enterobacterales (CPE). Objectives To assess the in vitro activity of imipenem/relebactam against a Spanish nationwide collection of CPE by testing the susceptibility of these isolates to 16 widely used antimicrobials and to determine the underlying β-lactam resistance mechanisms involved and the molecular epidemiology of carbapenemases in Spain. Materials and methods Clinical CPE isolates (n = 401) collected for 2 months from 24 hospitals in Spain were tested. MIC50, MIC90 and susceptibility/resistance rates were interpreted in accordance with the EUCAST guidelines. β-Lactam resistance mechanisms and molecular epidemiology were characterized by WGS. Results For all isolates, high rates of susceptibility to colistin (86.5%; MIC50/90 = 0.12/8 mg/L), imipenem/relebactam (85.8%; MIC50/90 = 0.5/4 mg/L) and ceftazidime/avibactam (83.8%, MIC50/90 = 1/≥256 mg/L) were observed. The subgroups of isolates producing OXA-48-like (n = 305, 75.1%) and KPC-like enzymes (n = 44, 10.8%) were highly susceptible to ceftazidime/avibactam (97.7%, MIC50/90 = 1/2 mg/L) and imipenem/relebactam (100.0%, MIC50/90 = ≤0.25/1 mg/L), respectively. The most widely disseminated high-risk clones of carbapenemase-producing Klebsiella pneumoniae across Spain were found to be ST11, ST147, ST392 and ST15 (mostly associated with OXA-48) and ST258/512 (in all cases producing KPC). Conclusions Imipenem/relebactam, colistin and ceftazidime/avibactam were the most active antimicrobials against all CPEs. Imipenem/relebactam is a valuable addition to the antimicrobial arsenal used in the fight against CPE, particularly against KPC-producing isolates, which in all cases were susceptible to this combination.

Control of infections caused by carbapenem-resistant Klebsiella pneumoniae continues to be challenging. The success of this pathogen is favored by its ability to acquire antimicrobial resistance and to spread and persist in both the environment and in humans.The emergence of clinically important clones, such as sequence types 11, 15, 101, and 258, has been reported worldwide. However,the mechanisms promoting the dissemination of such highrisk clones are unknown. Unraveling the factors that play a role in the pathobiology and epidemicity of K. pneumoniae is therefore important for managing infections. To address this issue, we studied a carbapenem-resistant ST-15 K. pneumoniae isolate (Kp3380) that displayed a remarkable adherent phenotype with abundant pilus-like structures. Genome sequencing enabled us to identify a chaperone-usher pili system (Kpi) in Kp3380. Analysis of a large K. pneumoniae population from 32 European countries showed that the Kpi system is associated with the ST-15 clone. Phylogenetic analysis of the operon revealed that Kpi belongs to the littlecharacterized γ2-fimbrial clade. We demonstrate that Kpi contributes positively to the ability of K.pneumoniae to form biofilms and adhere to different host tissues. Moreover, the in vivo intestinal colonizing capacity of the Kpi-defective mutant was significantly reduced, as was its ability to infect Galleria mellonella. The findings provide information about the pathobiology and epidemicity of Kpi+ K. pneumoniae and indicate that the presence of Kpi may explain the success of the ST-15 clone. Disrupting bacterial adherence to the intestinal surface could potentially target gastrointestinal colonization., This research was supported by Projects p-01216A and IJCI-2016-29524 (to A.P.), funded by the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) and the Minestry of Economy and Competetiveness(MINECO), respectively. It was also supported by Projects PI11/01034 (to M.P.), PI14/00059 and PI17/1482 (to M.P. and A.B.), and PI18/00501 (to G.B.),included in the National Plan for Scientific Research, Development and Technological Innovation 2013-2016 and funded by the Instituto de Salud Carlos III (ISCIII) and Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/006) cofinanced by European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth 2014-2020. Grant BFU2016-77835-R of the MINECO (to A.R.) also supported this research. E.G. was financially supported by the SEIMC project. J.C.V.-U. was financially supported by the PFIS (Contratos Predoctorales de Formación en Investigación en Salud) program (F18/00315);J.A.V. was financially supported by IN607A 2016/22; M.M.-G. was financially supported by a Clara Roy grant (SEIMC); A.B. was financially supported by the Miguel Servet program (ISCIII, Spain); B.K.R.-J. was financially supported by Marie S. Curie Action SaPhaDe project (MSCA-IF-GF-836754); and A.P. was financially supported by the Juan de la Cierva program (MINECO, IJCI-2016-29524).

Background Infections caused by multidrug-resistant pathogens such as Acinetobacter baumannii constitute a major health problem worldwide. In this study we present a global in vivo transcriptomic analysis of A. baumannii isolated from the lungs of mice with pneumonia infection. Methods Mice were infected with A. baumannii ATCC 17978 and AbH12O-A2 strains and the total bacterial RNA were analyzed by RNA sequencing. Lists of differentially expressed genes were obtained and 14 of them were selected for gene deletion and further analysis. Results Transcriptomic analysis revealed a specific gene expression profile in A. baumannii during lung infection with upregulation of genes involved in iron acquisition and host invasion. Mutant strains lacking feoA, mtnN, yfgC, basB, hisF, oatA, and bfnL showed a significant loss of virulence in murine pneumonia. A decrease in biofilm formation, adherence to human epithelial cells, and growth rate was observed in selected mutants. Conclusions This study provides an insight into A. baumannii gene expression profile during murine pneumonia infection. Data revealed that 7 in vivo upregulated genes were involved in virulence and could be considered new therapeutic targets.

The ability of 6-(aryl)methylidene penicillin-based sulfones 1-7 to repurpose β-lactam antibiotics activity with bacterial species that carry carbapenem-hydrolyzing class D β-lactamases (OXA-23, OXA-24/40 and OXA-48), as well as with class A (TEM-1, CTX-M-2) and class C (CMY-2, DHA-1) enzymes, is reported. The combinations imipenem/3 and imipenem/4 restored almost completely the antibiotic efficacy in OXA-23 and OXA-24/40 carbapenemase-producing A. baumannii strains (1 μg mL-1) and also provided good results for OXA-48 carbapenemase-producing K. pneumoniae strains (4 μg mL-1). Compounds 2-6 in combinations with ceftazidime and ampicillin were also efficient in restoring antibiotic efficacy in E. coli strains carrying class C (CMY-2 and DHA-1) and class A (TEM-1 and CTX-M-2) β-lactamase enzymes, respectively. Kinetic and inhibition studies with the OXA-24/40 enzyme, protein mass spectrometry analysis and docking studies allowed us to gain an insight into the inhibition mechanism and the experimentally observed differences between the ligands.

The pyrrole-imidazoles, a group of alkaloids commonly found in marine sponges belonging to the genus Agelas, display a wide range of biological activities. Herein, we report the first chemical study of the secondary metabolites of the sponge A. dilatata from the coastal area of the Yucatan Peninsula (Mexico). In this study, we isolated eight known alkaloids from an organic extract of the sponge. We used NMR and MS analysis and comparison with existing databases to characterize the alkaloids: ageliferin (1), bromoageliferin (2), dibromoageliferin (3), sceptrin (4), nakamuric acid (5), 4-bromo-1H-pyrrole-2-carboxylic acid (6), 4,5-dibromopyrrole-2-carboxylic acid (7) and 3,7-dimethylisoguanine (8). We also evaluated, for the first time, the activity of these alkaloids against the most problematic multidrug-resistant (MDR) pathogens, i.e., the Gram-negative bacteria Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii. Bromoageliferin (2) displayed significant activity against P. aeruginosa. Comparison of the antibacterial activity of ageliferins 1&ndash, 3 (of similar structure) against P. aeruginosa revealed some relationship between structure and activity. Furthermore, in in vitro assays, 2 inhibited growth and biofilm production in clinical strains of P. aeruginosa. Moreover, 2 increased the survival time in an in vivo Galleria mellonella model of infection. The findings confirm bromoageliferin (2) as a potential lead for designing new antibacterial drugs.

Selection of extended-spectrum mutations in narrow-spectrum oxacillinases (e.g., OXA-2 and OXA-10) is an emerging mechanism for development of in vivo resistance to ceftolozane-tazobactam and ceftazidime-avibactam in Pseudomonas aeruginosa. Detection of these challenging enzymes therefore seems essential to prevent clinical failure, but the complex phenotypic plasticity exhibited by this species may often lead to their underestimation. The underlying resistance mechanisms of two sequence type 175 (ST175) P. aeruginosa isolates showing multidrug-resistant phenotypes and recovered at early and late stages of a long-term nosocomial infection were evaluated. Whole-genome sequencing (WGS) was used to investigate resistance genomics, whereas molecular and biochemical methods were used for characterization of a novel extended-spectrum OXA-2 variant selected during therapy. The metallo-β-lactamase bla(VIM-20) and the narrow-spectrum oxacillinase bla(OXA-2) were present in both isolates, although they differed by an inactivating mutation in the mexB subunit, present only in the early isolate, and in a mutation in the bla(OXA-2) β-lactamase, present only in the final isolate. The new OXA-2 variant, designated OXA-681, conferred elevated MICs of the novel cephalosporin–β-lactamase inhibitor combinations in a PAO1 background. Compared to OXA-2, kinetic parameters of the OXA-681 enzyme revealed a substantial increase in the hydrolysis of cephalosporins, including ceftolozane. We describe the emergence of the novel variant OXA-681 during treatment of a nosocomial infection caused by a Pseudomonas aeruginosa ST175 high-risk clone. The ability of OXA-681 to confer cross-resistance to ceftolozane-tazobactam and ceftazidime-avibactam together with the complex antimicrobial resistance profiles exhibited by the clinical strains harboring this new enzyme argue for maintaining active surveillance on emerging broad-spectrum resistance in P. aeruginosa.

The carbapenem-hydrolyzing class D β-lactamases (CHDLs) are the main mechanism of carbapenem resistance in Acinetobacter baumannii. CHDLs are not effectively inactivated by clinically available β-lactam-type inhibitors. We have previously described the in vitro efficacy of the inhibitor LN-1-255 in combination with carbapenems. The aim of this study was to compare the efficacy of LN-1-255 with that of imipenem in murine pneumonia using A. baumannii strains carrying their most extended carbapenemases, OXA-23 and OXA-24/40. The bla(OXA-23) and bla(OXA-24/40) genes were cloned into the carbapenem-susceptible A. baumannii ATCC 17978 strain. Clinical isolates Ab1 and JC12/04, producing the enzymes OXA-23 and OXA-24/40, respectively, were used in the study. Pharmacokinetic (PK) parameters were determined. An experimental pneumonia model was used to evaluate the efficacy of the combined imipenem–LN-1-255 therapy. MICs of imipenem decreased between 32- and 128-fold in the presence of LN-1-255. Intramuscular treatment with imipenem–LN-1-255 (30/50 mg/kg) decreased the bacterial burden by (i) 4 and 1.7 log(10) CFU/g lung in the infection with the ATCC 17978-OXA-23 and Ab1 strains, respectively, and by (ii) 2.5 and 4.5 log(10) CFU/g lung in the infection produced by the ATCC 17978-OXA-24/40 and the JC12/04 strains, respectively. In all assays, combined therapy offered higher protection against pneumonia than that provided by monotherapy. No toxicity was observed in treated mice. Imipenem treatment combined with LN-1-255 treatment significantly reduced the severity of infection by carbapenem-resistant A. baumannii strains carrying CHDLs. Preclinical assays demonstrated the potential of LN-1-255 and imipenem therapy as a new antibacterial treatment.

Acinetobacter baumannii is currently considered one of the most problematic nosocomial microorganisms. In the present work the hisF gene from the ATCC 17978 strain and the AbH12O-A2 clinical isolate of A. baumannii was found over-expressed during the course of murine pneumonia infections. The study demonstrated that the A. baumannii ATCC 17978 mutant strain lacking the hisF gene induces a sub-lethal pneumonia infection in mice, while the complemented mutant strain increased its virulence. This histidine auxotroph mutant showed an increase on IL-6 secretion and leukocytes recruitment during infections. Furthermore, data revealed that the hisF gene, implicated in the innate immunity and inflammation, is involved in virulence during a pneumonia infection, which may partly explain the ability of this strain to persist in the lung. We suggest that HisF, essential for full virulence in this pathogen, should be considered a potential target for developing new antimicrobial therapies against A. baumannii.Importance Nosocomial pathogens such as A. baumannii are able to acquire and develop multi-drug resistance and represent an important clinical and economic problem. There is therefore an urgent need to find new therapeutic targets to fight against A. baumannii. In the present work, the potential of HisF from A. baumannii as a therapeutic target has been addressed since this protein is involved in the innate inmunity and the inflamatory response and seems essential to develop a pneumonia in mice. This work lays the groundwork for designing antimicrobial therapies that block the activity of HisF.

ThehisFgene fromA. baumanniiATCC 17978 was found over-expressed during a murine pneumonia infection. A mutant strain lackinghisFshowed its involvement in virulence during mice pneumonia as well as in host inflammatory response, where the product of HisF may act as negative regulator in the production of pro-inflammatory cytokines. This work evaluates the role of HisF in theA. baumanniipathogenesis and suggests its potential as a new target for antimicrobial therapies.

This is a pre-copyedited, author-produced version of an article accepted for publication in Journal of Antimicrobial Chemotherapy following peer review. The version of record Juan A. Vallejo, Marta Martínez-Guitián, Juan C. Vázquez-Ucha, Concepción González-Bello, Margarita Poza, John D. Buynak, Christopher R. Bethel, Robert A. Bonomo, German Bou, Alejandro Beceiro; LN-1-255, a penicillanic acid sulfone able to inhibit the class D carbapenemase OXA-48, Journal of Antimicrobial Chemotherapy, Volume 71, Issue 8, 1 August 2016, Pages 2171–2180 is available online at: https://doi.org/10.1093/jac/dkw105 Objectives Carbapenemases are the most important mechanism responsible for carbapenem resistance in Enterobacteriaceae. Among carbapenemases, OXA-48 presents unique challenges as it is resistant to β-lactam inhibitors. Here, we test the capacity of the compound LN-1-255, a 6-alkylidene-2′-substituted penicillanic acid sulfone, to inhibit the activity of the carbapenemase OXA-48. Methods The OXA-48 gene was cloned and expressed in Klebsiella pneumoniae and Escherichia coli in order to obtain MICs in the presence of inhibitors (clavulanic acid, tazobactam and sulbactam) and LN-1-255. OXA-48 was purified and steady-state kinetics was performed with LN-1-255 and tazobactam. The covalent binding mode of LN-1-255 with OXA-48 was studied by docking assays. Results Both OXA-48-producing clinical and transformant strains displayed increased susceptibility to carbapenem antibiotics in the presence of 4 mg/L LN-1-255 (2–32-fold increased susceptibility) and 16 mg/L LN-1-255 (4–64-fold increased susceptibility). Kinetic assays demonstrated that LN-1-255 is able to inhibit OXA-48 with an acylation efficiency (k2/K) of 10 ± 1 × 104 M−1 s−1 and a slow deacylation rate (koff) of 7 ± 1 × 10−4 s−1. IC50 was 3 nM for LN-1-255 and 1.5 μM for tazobactam. Lastly, kcat/kinact was 500-fold lower for LN-1-255 than for tazobactam. Conclusions In these studies, carbapenem antibiotics used in combination with LN-1-255 are effective against the carbapenemase OXA-48, an important emerging mechanism of antibiotic resistance. This provides an incentive for further investigations to maximize the efficacy of penicillin sulfone inhibition of class D plasmid-carried Enterobacteriaceae carbapenemases. This work was supported by the Spanish National Plans for Scientific Research, Development and Technological Innovation 2013-16 and funded by the ISCIII-General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (ERDF) ‘A way of making Europe’: PI12/00552 to G. B. and PI14/00059 to M. P. and A. B. Also, research reported in this publication was supported in part by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (USA) under Award Numbers R01AI100560, R01AI063517 and R01AI072219 to R. A. B. This study was supported in part by funds and/or facilities provided by the Cleveland Department of Veterans Affairs (USA), Award Number 1I01BX001974 to R. A. B. from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development and the Geriatric Research Education and Clinical Center VISN 10 (USA) to R. A. B. This study was also supported by the Spanish Ministry of Economy and Competiveness (SAF2013-42899-R), Xunta de Galicia (Spain) (GRC2013-041) and the European Regional Development Fund (ERDF) to C. G.-B, and supported by National Institutes of Health (USA) to J. D. B. (1R15AI109624). J. V. A. was financially supported by the Sara Borrell Programme ISCIII-FEDER (CD13/00373). J. V. H. and A. B. were financially supported by the Miguel Servet Programme ISCIII-FEDER (CP13/00226) SI

Acinetobacter baumannii is a nosocomial pathogen that has a considerable ability to survive in the hospital environment partly due to its capacity to form biofilms. The first step in the process of establishing an infection is adherence of the bacteria to target cells. Chaperone-usher pili assembly systems are involved in pilus biogenesis pathways that play an important role in adhesion to host cells and tissues as well as medically relevant surfaces. After screening a collection of strains, a biofilm hyper-producing A. baumannii strain (MAR002) was selected to describe potential targets involved in pathogenicity. MAR002 showed a remarkable ability to form biofilm and attach to A549 human alveolar epithelial cells. Analysis of MAR002 using transmission electron microscopy (TEM) showed a significant presence of pili on the bacterial surface. Putative protein-coding genes involved in pili formation were identified based on the newly sequenced genome of MAR002 strain (JRHB01000001/2 or NZ_JRHB01000001/2). As assessed by qRT-PCR, the gene LH92_11085, belonging to the operon LH92_11070-11085, is overexpressed (ca. 25-fold more) in biofilm-associated cells compared to exponential planktonic cells. In the present work we investigate the role of this gene on the MAR002 biofilm phenotype. Scanning electron microscopy (SEM) and biofilm assays showed that inactivation of LH92_11085 gene significantly reduced bacterial attachment to A549 cells and biofilm formation on plastic, respectively. TEM analysis of the LH92_11085 mutant showed the absence of long pili formations normally present in the wild-type. These observations indicate the potential role this LH92_11085 gene could play in the pathobiology of A baumannii., The results of this work have been funded by the Projects PI11/01034 to MP and PI14/00059 to MP and AB and PI12/00552 and PI15/00860 to GB, integrated in the National Plan for Scientific Research, Development and Technological Innovation 2013-2016 and funded by the ISCIII- General Subdirection of Assessment and Promotion of the Research – European Regional Development Fund (FEDER) “A way of making Europe” and Miami University (Ohio, USA) Research Funds. We also thank Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0014 to GB), cofinanced by the European Development Regional Fund (EDRF) “A Way to Achieve Europe, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad. A.P. was financially supported by the Galician Plan for Research, Innovation and Growth (I2C Plan 2012-2016). J.A. Vallejo was financially supported by the Sara Borrell Program (ISCIII, Spain CD13/00373). We wish to thank grant GEIH/REIPI-Ab2010 from Instituto de Salud Carlos III - Ministerio Economía y Competitividad for providing MAR002 strain and M.I. Voskuil for pMo130.

[Abstract] A total of 51 sponges (Porifera) and 13 ascidians (Chordata) were collected on the coast of the Yucatan Peninsula (Mexico) and extracted with organic solvents. The resulting extracts were screened for antibacterial activity against four multidrug-resistant (MDR) bacterial pathogens: the Gram-negative Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus. The minimum inhibitory concentrations (MICs) of the organic extracts of each marine organism were determined using a broth microdilution assay. Extracts of eight of the species, in particular the Agelas citrina and Haliclona (Rhizoniera) curacaoensis, displayed activity against some of the pathogens tested. Some of the extracts showed similar MIC values to known antibiotics such as penicillins and aminoglycosides. This study is the first to carry out antimicrobial screening of extracts of marine sponges and ascidians collected from the Yucatan Peninsula. Bioassay-guided fractionation of the active extracts from the sponges Amphimedon compressa and A. citrina displayed, as a preliminary result, that an inseparable mixture of halitoxins and amphitoxins and (-)-agelasine B, respectively, are the major compounds responsible for their corresponding antibacterial activities. This is the first report of the antimicrobial activity of halitoxins and amphitoxins against major multidrug-resistant human pathogens. The promising antibacterial activities detected in this study indicate the coast of Yucatan Peninsula as a potential source of a great variety of marine organisms worthy of further research. This work was supported by Grants RTI2018-093634-B-C22 (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, co-funded by the FEDER Programme from the European Union, by projects PI15/00860 and PI18/00501 to GB and PI14/00059 and PI17/01482 to AB (Instituto de Salud Carlos III) and BLUEBIOLAB (0474_BLUEBIOLAB_1_E), Programme INTERREG V A of Spain-Portugal (POCTEP). The study was also funded by projects IN607A 2016/22 (GAIN- Agencia Gallega de Innovación - Consellería de Economía, Emprego e Industria) and GRC2018/039 and Agrupación Estratégica CICA-INIBIC ED431E 2018/03 (Consellería de Educación, Universidades e Formación Profesional) from the Xunta de Galicia (autonomous government of the region). Support was also provided by Planes Nacionales de I+D+i 2013-2016 and ISCIII, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/006) co-financed by European Development Regional Fund "A way to achieve Europe" and the operative program Intelligent Growth 2014-2020. J.C.V.U. was financially supported by the pFIS Programme (FI18/00315), M.M.G. was financially supported by a Clara Roy grant (SEIMC) and C.L.M. by IN606A-2019/029. D.P.P. received a fellowship from the program National Council of Science and Technology (CONACYT) of Mexico and the Secretariat of Research, Innovation and Higher Education (SIIES) of Yucatan (Mexico) Xunta de Galicia; 0474_BLUEBIOLAB_1_E Xunta de Galicia; IN607A 2016/22 Xunta de Galicia; GRC2018/039 Xunta de Galicia; ED431E 2018/03 Xunta de Galicia; IN606A-2019/029

The number of infections caused by Gram-negative pathogens carrying carbapenemases is increasing, and the group of carbapenem-hydrolyzing class D β-lactamases (CHDLs) is especially problematic. Several clinically important CHDLs have been identified in A. baumannii, including OXA-23, OXA-24/40, OXA-58, OXA-143, OXA-235, and the chromosomally encoded OXA-51. The selection and dissemination of carbapenem-resistant A. baumannii strains constitutes a serious global threat. Carbapenems have been successfully utilized as last resort antibiotics for the treatment of multi-drug-resistant A. baumannii infections. However, the spread of OXA carbapenemases is compromising the continued use of these antimicrobials. In response to this clinical issue, it is necessary and urgent to design and develop new specific inhibitors with efficacy against these enzymes. The aim of this work is to characterize the inhibitory activity of LN-1-255 (a 6-alkylidene-2-substituted penicillin sulfone) and compare it to that of two established inhibitors (avibactam and tazobactam) against the most relevant enzymes of each group of class D carbapenemases in A. baumannii. The β-lactamase inhibitor LN-1-255 demonstrated excellent microbiological synergy and inhibition kinetics parameters against all tested CHDLs, and a significantly higher activity than tazobactam and avibactam. A combination of carbapenems and LN-1-255 was effective against A. baumannii class D carbapenemases. Docking assays confirmed the affinity of LN-1-255 for the active site of these enzymes. LN-1-255 represents a potential new β-lactamase inhibitor, which may have a significant role in eradicating infections caused by A. baumannii isolates carrying CHDLs This work was supported by the Spanish National Plans for Scientific Research, Development and Technological Innovation 2008-2011 and 2013-2016 and funded by the ISCIII- General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (ERDF) “A way of making Europe”: PI12/00552 to G.B. and PI14/00059 to M.P. and A.B. Also, this study was supported in part by funds from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (USA) under award numbers R01AI063517 and R01AI100560, by funds and/or facilities provided by the Cleveland Department of Veterans Affairs, the Veterans Affairs Merit Review Program Award 1I01BX001974 and the Geriatric Research Education and Clinical Center VISN 10 to R.A.B., and by the Spanish Ministry of Economy and Competiveness (SAF2013-42899-R), Xunta de Galicia (GRC2013-041) and the European Regional Development Fund (ERDF) to C.GB. J.V. was financially supported by the Sara Borrell Programme ISCIII-FEDER (CD13/00373). J.V.H. and A.B. were financially supported by the Miguel Servet Programme ISCIII-FEDER (CP13/00226) SI

Synergy between colistin and the signal peptidase inhibitor MD3 was tested against isogenic mutants and clinical pairs of Acinetobacter baumannii isolates. Checkerboard assays and growth curves showed synergy against both colistin-susceptible strains (fractional inhibitory concentration index [FIC index ] = 0.13 to 0.24) and colistin-resistant strains with mutations in pmrB and phosphoethanolamine modification of lipid A (FIC index = 0.14 to 0.25) but not against colistin-resistant Δ lpx strains with loss of lipopolysaccharide (FIC index = 0.75 to 1). A colistin/MD3 combination would need to be targeted to strains with specific colistin resistance mechanisms.

The large conformational changes observed by Molecular Dynamics simulation studies on the product release in the LID and shikimic acid binding (SB) domains of the shikimate kinase (SK) enzyme have been exploited in the development of reversible competitive inhibitors against SK from Mycobacterium tuberculosis and Helicobacter pylori. This enzyme is a recognized target for antibiotic drug discovery. The reported C5-substituted shikimic acid analogues interact with the dynamic apolar pocket that surrounds the C4 and C5 hydroxyl groups of the natural substrate, cause the opening of the LID and SB domains, and capture the essential arginine far from the ATP binding site as required for catalysis. The 3-nitrobenzyl 3e and 5-benzothiophenyl derivatives 3i proved to be the most potent inhibitors. An ester prodrug of 3i was the most efficient derivative in achieving good in vitro activity against H. pylori, having a MIC value of 4 μg/mL Financial support from the Spanish Ministry of Economy and Competiveness (SAF2013-42899-R), Xunta de Galicia (GRC2013-041) and the European Regional Development Fund (ERDF) is gratefully acknowledged. V.P. and M.M. thank the Spanish Ministry of Economy and Competiveness and the Spanish Ministry of Education for their respective FPI and FPU fellowships. E.L. thanks the Xunta de Galicia for his postdoctoral fellowship. J.C.V.-U. and A.B. thank the Miguel Servet Programme ISCIII-FEDER (CP13/00226) and the ISCIII General Subdirection of Assesment and Promotion of the Research (PI14/00059) for financial support SI

BackgroundThe development of resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of Pseudomonas aeruginosa infections is concerning.ObjectivesCharacterization of the mechanisms leading to the development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR P. aeruginosa infections.MethodsFour paired ceftolozane/tazobactam- and ceftazidime/avibactam-susceptible/resistant isolates were evaluated. MICs were determined by broth microdilution. STs, resistance mechanisms and genetic context of β-lactamases were determined by genotypic methods, including WGS. The OXA-10 variants were cloned in PAO1 to assess their impact on resistance. Models for the OXA-10 derivatives were constructed to evaluate the structural impact of the amino acid changes.ResultsThe same XDR ST253 P. aeruginosa clone was detected in all four cases evaluated. All initial isolates showed OprD deficiency, produced an OXA-10 enzyme and were susceptible to ceftazidime, ceftolozane/tazobactam, ceftazidime/avibactam and colistin. During treatment, the isolates developed resistance to all cephalosporins. Comparative genomic analysis revealed that the evolved resistant isolates had acquired mutations in the OXA-10 enzyme: OXA-14 (Gly157Asp), OXA-794 (Trp154Cys), OXA-795 (ΔPhe153-Trp154) and OXA-824 (Asn143Lys). PAO1 transformants producing the evolved OXA-10 derivatives showed enhanced ceftolozane/tazobactam and ceftazidime/avibactam resistance but decreased meropenem MICs in a PAO1 background. Imipenem/relebactam retained activity against all strains. Homology models revealed important changes in regions adjacent to the active site of the OXA-10 enzyme. The blaOXA-10 gene was plasmid borne and acquired due to transposition of Tn6746 in the pHUPM plasmid scaffold.ConclusionsModification of OXA-10 is a mechanism involved in the in vivo acquisition of resistance to cephalosporin/β-lactamase inhibitor combinations in P. aeruginosa.