Epidemiology, resistance genomics and susceptibility of Acinetobacter species: results from the 2020 Spanish nationwide surveillance study.

BackgroundAs increasing antibiotic resistance in Acinetobacter baumannii poses a global healthcare challenge, understanding its evolution is crucial for effective control strategies.AimWe aimed to evaluate the epidemiology, antimicrobial susceptibility and main resistance mechanisms of Acinetobacter spp. in Spain in 2020, and to explore temporal trends of A. baumannii.MethodsWe collected 199 single-patient Acinetobacter spp. clinical isolates in 2020 from 18 Spanish tertiary hospitals. Minimum inhibitory concentrations (MICs) for nine antimicrobials were determined. Short-read sequencing was performed for all isolates, and targeted long-read sequencing for A. baumannii. Resistance mechanisms, phylogenetics and clonality were assessed. Findings on resistance rates and infection types were compared with data from 2000 and 2010.ResultsCefiderocol and colistin exhibited the highest activity against A. baumannii, although colistin susceptibility has significantly declined over 2 decades. A. non-baumannii strains were highly susceptible to most tested antibiotics. Of the A. baumannii isolates, 47.5% (56/118) were multidrug-resistant (MDR). Phylogeny and clonal relationship analysis of A. baumannii revealed five prevalent international clones, notably IC2 (ST2, n = 52; ST745, n = 4) and IC1 (ST1, n = 14), and some episodes of clonal dissemination. Genes bla OXA-23, bla OXA-58 and bla OXA-24/40 were identified in 49 (41.5%), eight (6.8%) and one (0.8%) A. baumannii isolates, respectively. ISAba1 was found upstream of the gene (a bla OXA-51-like) in 10 isolates.ConclusionsThe emergence of OXA-23-producing ST1 and ST2, the predominant MDR lineages, shows a pivotal shift in carbapenem-resistant A. baumannii (CRAB) epidemiology in Spain. Coupled with increased colistin resistance, these changes underscore notable alterations in regional antimicrobial resistance dynamics.

Antimicrobial Activity of Cefiderocol against the Carbapenemase-Producing Enterobacter cloacae Complex and Characterization of Reduced Susceptibility Associated with Metallo-ß-Lactamase VIM-1.

Emergence of cefiderocol resistance among carbapenemase-producing Enterobacterales, particularly those in the Enterobacter cloacae complex (ECC), is becoming of alarming concern; however, the mechanistic basis of this phenomenon remains poorly understood. We describe the acquisition of VIM-1-mediated reduced cefiderocol susceptibility (MICs 0.5 to 4 mg/L) in a collection of 54 carbapenemase-producing isolates belonging to the ECC. MICs were determined by reference methodologies. Antimicrobial resistance genomic analysis was performed through hybrid WGS. The impact of VIM-1 production on cefiderocol resistance in the ECC background was examined at microbiological, molecular, biochemical, and atomic levels. Antimicrobial susceptibility testing yielded 83.3% susceptible isolates and MIC50/90 values of 1/4 mg/L. Decreased susceptibility to cefiderocol was mainly associated with isolates producing VIM-1, with cefiderocol MICs 2- to 4-fold higher than for isolates carrying other types of carbapenemases. E. cloacae and Escherichia coli VIM-1 transformants displayed significantly enhanced cefiderocol MICs. Biochemical assays with purified VIM-1 protein revealed low but detectable cefiderocol hydrolysis. Simulation studies revealed how cefiderocol is anchored to the VIM-1 active site. Additional molecular assays and WGS data analysis highlighted the implication of SHV-12 coproduction and suggested the inactivation of the FcuA-like siderophore receptor as further contributors to the higher cefiderocol MICs. Our findings warn of the potential of the VIM-1 carbapenemase to at least partly limit the activity of cefiderocol in the ECC. This effect is probably enhanced due to combination with additional mechanisms, such as ESBL production and siderophore inactivation, and indicates the need for active surveillance to extend the life span of this promising cephalosporin.

Direct Detection of Carbapenemase-Producing Klebsiella pneumoniae by MALDI-TOF Analysis of Full Spectra Applying Machine Learning.

MALDI-TOF MS is considered to be an important tool for the future development of rapid microbiological techniques. We propose the application of MALDI-TOF MS as a dual technique for the identification of bacteria and the detection of resistance, with no extra hands-on procedures. We have developed a machine learning approach that uses the random forest algorithm for the direct prediction of carbapenemase-producing Klebsiella pneumoniae (CPK) isolates, based on the spectra of complete cells. For this purpose, we used a database of 4,547 mass spectra profiles, including 715 unduplicated clinical isolates that are represented by 324 CPK with 37 different ST. The impact of the culture medium was determinant in the CPK prediction, being that the isolates were tested and cultured in the same media, compared to the isolates used to build the model (blood agar). The proposed method has an accuracy of 97.83% for the prediction of CPK and an accuracy of 95.24% for the prediction of OXA-48 or KPC carriage. For the CPK prediction, the RF algorithm yielded a value of 1.00 for both the area under the receiver operating characteristic curve and the area under the precision-recall curve. The contribution of individual mass peaks to the CPK prediction was determined using Shapley values, which revealed that the complete proteome, rather than a series of mass peaks or potential biomarkers (as previously suggested), is responsible for the algorithm-based classification. Thus, the use of the full spectrum, as proposed here, with a pattern-matching analytical algorithm produced the best outcome. The use of MALDI-TOF MS coupled with machine learning algorithm processing enabled the identification of CPK isolates within only a few minutes, thereby reducing the time to detection of resistance.

Simultaneous and divergent evolution of resistance to cephalosporin/ß-lactamase inhibitor combinations and imipenem/relebactam following ceftazidime/avibactam treatment of MDR Pseudomonas aeruginosa infections.

OBJECTIVES: To describe and characterize the emergence of resistance to ceftolozane/tazobactam, ceftazidime/avibactam and imipenem/relebactam in a patient receiving ceftazidime/avibactam treatment for an MDR Pseudomonas aeruginosa CNS infection. METHODS: One baseline (PA1) and two post-exposure (PA2 and PA3) isolates obtained before and during treatment of a nosocomial P. aeruginosa meningoventriculitis were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. The impact on ß-lactam resistance of mutations in blaPDC and mexR was determined through cloning experiments and complementation assays. RESULTS: Isolate PA1 showed baseline resistance mutations in DacB (I354A) and OprD (N142fs) conferring resistance to conventional antipseudomonals but susceptibility to ceftazidime/avibactam, ceftolozane/tazobactam and imipenem/relebactam. Post-exposure isolates showed two divergent ceftazidime/avibactam-resistant phenotypes associated with distinctive mutations affecting the intrinsic P PDC ß-lactamase (S254Ins) (PA2: ceftolozane/tazobactam and ceftazidime/avibactam-resistant) or MexAB-OprM negative regulator MexR in combination with modification of PBP3 (PA3: ceftazidime/avibactam and imipenem/relebactam-relebactam-resistant). Cloning experiments demonstrated the role of PDC modification in resistance to ceftolozane/tazobactam and ceftazidime/avibactam. Complementation with a functional copy of the mexR gene in isolate PA3 restored imipenem/relebactam susceptibility. CONCLUSIONS: We demonstrated how P. aeruginosa may simultaneously develop resistance and compromise the activity of new ß-lactam/ß-lactamase inhibitor combinations when exposed to ceftazidime/avibactam through selection of mutations leading to PDC modification and up-regulation of MexAB-OprM-mediated efflux.

Kpi, a chaperone-usher pili system associated with the worldwide-disseminated high-risk clone Klebsiella pneumoniae ST-15.

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 high-risk 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 little-characterized 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.

Assessment of Activity and Resistance Mechanisms to Cefepime in Combination with the Novel ß-Lactamase Inhibitors Zidebactam, Taniborbactam, and Enmetazobactam against a Multicenter Collection of Carbapenemase-Producing Enterobacterales.

The global distribution of carbapenemases such as KPC, OXA-48, and metallo-ß-lactamases (MBLs) gives cause for concern, as these enzymes are not inhibited by classical ß-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 (MIC50/90 ≤0.5/1 and ≤0.5/2 mg/L, respectively). Cefepime/zidebactam displayed high activity, regardless of the carbapenemase or extended-spectrum ß-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 (MIC50/90 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 ß-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.

Selection of AmpC ß-Lactamase Variants and Metallo-ß-Lactamases Leading to Ceftolozane/Tazobactam and Ceftazidime/Avibactam Resistance during Treatment of MDR/XDR Pseudomonas aeruginosa Infections.

Infections caused by ceftolozane-tazobactam and ceftazidime-avibactam-resistant P. aeruginosa infections are an emerging concern. We aimed to analyze the underlying ceftolozane-tazobactam and ceftazidime-avibactam resistance mechanisms in all multidrug-resistant or extensively drug-resistant (MDR/XDR) P. aeruginosa isolates recovered during 1 year (2020) from patients with a documented P. aeruginosa infection. Fifteen isolates showing ceftolozane-tazobactam and ceftazidime-avibactam resistance were evaluated. Clinical conditions, previous positive cultures, and ß-lactams received in the previous month were reviewed for each patient. MICs were determined by broth microdilution. Multilocus sequence types (MLSTs) and resistance mechanisms were determined using short- and long-read whole-genome sequencing (WGS). The impact of Pseudomonas-derived cephalosporinases (PDCs) on ß-lactam resistance was demonstrated by cloning into an ampC-deficient PAO1 derivative (PAOΔC) and construction of 3D models. Genetic support of acquired ß-lactamases was determined in silico from high-quality hybrid assemblies. In most cases, the isolates were recovered after treatment with ceftolozane-tazobactam or ceftazidime-avibactam. Seven isolates from different sequence types (STs) owed their ß-lactam resistance to chromosomal mutations and all displayed specific substitutions in PDC: Phe121Leu and Gly222Ser, Pro154Leu, Ala201Thr, Gly214Arg, ΔGly203-Glu219, and Glu219Lys. In the other eight isolates, the ST175 clone was overrepresented (6 isolates) and associated with IMP-28 and IMP-13, whereas two ST1284 isolates produced VIM-2. The cloned PDCs conferred enhanced cephalosporin resistance. The 3D PDC models revealed rearrangements affecting residues involved in cephalosporin hydrolysis. Carbapenemases were chromosomal (VIM-2) or plasmid-borne (IMP-28, IMP-13) and associated with class-1 integrons located in Tn402-like transposition modules. Our findings highlighted that cephalosporin/ß-lactamase inhibitors are potential selectors of MDR/XDR P. aeruginosa strains producing PDC variants or metallo-ß-lactamases. Judicious use of these agents is encouraged.

Activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam against ceftolozane/tazobactam- and ceftazidime/avibactam-resistant Pseudomonas aeruginosa.

OBJECTIVES: To evaluate the activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam against a clinical and laboratory collection of ceftolozane/tazobactam- and ceftazidime/avibactam-resistant Pseudomonas aeruginosa ß-lactamase mutants. METHODS: The activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam, cefepime/zidebactam and comparators was evaluated against a collection of 30 molecularly characterized ceftolozane/tazobactam- and/or ceftazidime/avibactam-resistant P. aeruginosa isolates from patients previously treated with cephalosporins. To evaluate how the different ß-lactamases in the clinical isolates affected the resistance to these agents, a copy of each blaPDC, blaOXA-2 and blaOXA-10 ancestral and mutant allele from the clinical isolates was cloned in pUCp24 and expressed in dual blaPDC-oprD (for blaPDC-like genes) or single oprD (for blaOXA-2-like and blaOXA-10-like genes) PAO1 knockout mutants. MICs were determined using reference methodologies. RESULTS: For all isolates, MICs were higher than 4 and/or 8 mg/L for ceftolozane/tazobactam and ceftazidime/avibactam, respectively. Cefiderocol was the most active agent, showing activity against all isolates, except one clinical isolate that carried an R504C substitution in PBP3 (MIC = 16 mg/L). Imipenem/relebactam was highly active against all isolates, except two clinical isolates that carried the VIM-20 carbapenemase. Cefepime/zidebactam and cefepime/taniborbactam displayed activity against most of the isolates, but resistance was observed in some strains with PBP3 amino acid substitutions or that overexpressed mexAB-oprM or mexXY efflux pumps. Evaluation of transformants revealed that OXA-2 and OXA-10 extended-spectrum variants cause a 2-fold increase in the MIC of cefiderocol relative to parental enzymes. CONCLUSIONS: Cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam show promising and complementary in vitro activity against ceftolozane/tazobactam- and ceftazidime/avibactam-resistant P. aeruginosa. These agents may represent potential therapeutic options for ceftolozane/tazobactam- and ceftazidime/avibactam-resistant P. aeruginosa infections.

Antimicrobial Diterpene Alkaloids from an Agelas citrina Sponge Collected in the Yucatán Peninsula.

Three new diterpene alkaloids, (+)-8-epiagelasine T (1), (+)-10-epiagelasine B (2), and (+)-12-hydroxyagelasidine C (3), along with three known compounds, (+)-ent-agelasine F (4), (+)-agelasine B (5), and (+)-agelasidine C (6), were isolated from the sponge Agelas citrina, collected on the coasts of the Yucatán Peninsula (Mexico). Their chemical structures were elucidated by 1D and 2D NMR spectroscopy, HRESIMS techniques, and a comparison with literature data. Although the synthesis of (+)-ent-agelasine F (4) has been previously reported, this is the first time that it was isolated as a natural product. The evaluation of the antimicrobial activity against the Gram-positive pathogens Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis showed that all of them were active, with (+)-10-epiagelasine B (2) being the most active compound with an MIC in the range of 1-8 µg/mL. On the other hand, the Gram-negative pathogenes Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae were also evaluated, and only (+)-agelasine B (5) showed a moderate antibacterial activity with a MIC value of 16 µg/mL.

Global Transcriptomic Analysis During Murine Pneumonia Infection Reveals New Virulence Factors in Acinetobacter baumannii.

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.

Molecular mechanisms driving the in vivo development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR Pseudomonas aeruginosa infections.

BACKGROUND: The development of resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of Pseudomonas aeruginosa infections is concerning. OBJECTIVES: Characterization 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. METHODS: Four paired ceftolozane/tazobactam- and ceftazidime/avibactam-susceptible/resistant isolates were evaluated. MICs were determined by broth microdilution. STs, resistance mechanisms and genetic context of ß-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. RESULTS: The 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. CONCLUSIONS: Modification of OXA-10 is a mechanism involved in the in vivo acquisition of resistance to cephalosporin/ß-lactamase inhibitor combinations in P. aeruginosa.

Activity of imipenem/relebactam against a Spanish nationwide collection of carbapenemase-producing Enterobacterales.

BACKGROUND: Imipenem/relebactam is a novel carbapenem/ß-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 ß-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. ß-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.

Antisense inhibition of lpxB gene expression in Acinetobacter baumannii by peptide-PNA conjugates and synergy with colistin.

BACKGROUND: LpxB is an enzyme involved in the biosynthesis pathway of lipid A, a component of LPS. OBJECTIVES: To 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. METHODS: RNA-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. RESULTS: The 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. CONCLUSIONS: The 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.

Molecular and biochemical insights into the in vivo evolution of AmpC-mediated resistance to ceftolozane/tazobactam during treatment of an MDR Pseudomonas aeruginosa infection.

BACKGROUND: Pseudomonas aeruginosa may develop resistance to novel cephalosporin/ß-lactamase inhibitor combinations during therapy through the acquisition of structural mutations in AmpC. OBJECTIVES: To 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. METHODS: Paired 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. RESULTS: WGS 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. CONCLUSIONS: We 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/ß-lactamase inhibitors in P. aeruginosa.

In Vitro and In Vivo Assessment of the Efficacy of Bromoageliferin, an Alkaloid Isolated from the Sponge Agelas dilatata, against Pseudomonas aeruginosa.

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 1-3 (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.

Marine Organisms from the Yucatan Peninsula (Mexico) as a Potential Natural Source of Antibacterial Compounds.

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.

New Carbapenemase Inhibitors: Clearing the Way for the ß-Lactams.

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 ß-lactam/ß-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-ß-lactamases and carbapenem-hydrolysing class D ß-lactamases, not currently inhibited by any clinically approved inhibitor.

Challenging Antimicrobial Susceptibility and Evolution of Resistance (OXA-681) during Treatment of a Long-Term Nosocomial Infection Caused by a Pseudomonas aeruginosa ST175 Clone.

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-ß-lactamase blaVIM-20 and the narrow-spectrum oxacillinase blaOXA-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 blaOXA-2 ß-lactamase, present only in the final isolate. The new OXA-2 variant, designated OXA-681, conferred elevated MICs of the novel cephalosporin-ß-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.

Therapeutic Efficacy of LN-1-255 in Combination with Imipenem in Severe Infection Caused by Carbapenem-Resistant Acinetobacter baumannii.

The carbapenem-hydrolyzing class D ß-lactamases (CHDLs) are the main mechanism of carbapenem resistance in Acinetobacter baumannii CHDLs are not effectively inactivated by clinically available ß-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 blaOXA-23 and blaOXA-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 log10 CFU/g lung in the infection with the ATCC 17978-OXA-23 and Ab1 strains, respectively, and by (ii) 2.5 and 4.5 log10 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.