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The Journal of Biological Chemistry Jun 2024Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an...
Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain of SulM, which catalyzes an unusual β-lactam forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the monobactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstream holo peptidyl-carrier domain. The position of variant lid helices results in an active site pocket that is quite constrained, a feature that is likely necessary to orient the substrate properly for β-lactam formation. Modeling of a sulfazecin tripeptide into the active site identifies a plausible binding mode identifying potential interactions for the sulfamate and the peptide backbone with Arg2849 and Asn2819, respectively. The overall structure is similar to the β-lactone forming thioesterase domain that is responsible for similar ring closure in the production of obafluorin. We further use these insights to enable bioinformatic analysis to identify additional, uncharacterized β-lactam-forming biosynthetic gene clusters by genome mining.
PubMed: 38908753
DOI: 10.1016/j.jbc.2024.107489 -
BMJ Case Reports Jun 2024A man in his 40s with type 2 diabetes mellitus had persistent right-sided watery nasal discharge for 6 months following cerebrospinal fluid (CSF) leak repair at...
A man in his 40s with type 2 diabetes mellitus had persistent right-sided watery nasal discharge for 6 months following cerebrospinal fluid (CSF) leak repair at another hospital, prompting his visit to us due to recurring symptoms. Imaging revealed a CSF leak from the mid-clivus for which revision endoscopic CSF leak repair was done. Regrettably, he developed postoperative meningitis caused by multidrug-resistant (MDR) Managing this complex case was a challenging task due to the pathogen's resistance to conventional drugs and the scarcity of scientific evidence. We initiated a culture-guided combination regimen with ceftazidime, avibactam, aztreonam and tigecycline. This decision stemmed from meticulous literature review and observed antibiotic synergy while testing for this organism.After 4 weeks of vigilant treatment, the patient's symptoms improved significantly, and CSF cultures were sterile. We present our approach to effectively confront and manage a challenging instance of postoperative MDR bacterial meningitis.
Topics: Humans; Male; Klebsiella Infections; Meningitis, Bacterial; Anti-Bacterial Agents; Klebsiella pneumoniae; Drug Resistance, Multiple, Bacterial; Cerebrospinal Fluid Leak; Adult; Postoperative Complications; Ceftazidime; Cranial Fossa, Posterior; Aztreonam; Tigecycline; Drug Combinations; Azabicyclo Compounds
PubMed: 38885998
DOI: 10.1136/bcr-2023-257872 -
Annals of Clinical Microbiology and... May 2024Aztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility... (Comparative Study)
Comparative Study
BACKGROUND
Aztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches.
METHODS
96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher).
RESULTS
According to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%).
CONCLUSIONS
The ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.
Topics: Aztreonam; Azabicyclo Compounds; Microbial Sensitivity Tests; Anti-Bacterial Agents; Humans; Drug Resistance, Multiple, Bacterial; Gram-Negative Bacteria; Drug Combinations; Pseudomonas aeruginosa; beta-Lactamases; Enterobacteriaceae; Bacterial Proteins; Gram-Negative Bacterial Infections
PubMed: 38796461
DOI: 10.1186/s12941-024-00708-0 -
Antibiotics (Basel, Switzerland) Apr 2024Patients hospitalized in ICUs with severe COVID-19 are at risk for developing hospital-acquired infections, especially infections caused by . We aimed to describe the...
Patients hospitalized in ICUs with severe COVID-19 are at risk for developing hospital-acquired infections, especially infections caused by . We aimed to describe the evolution of infections in ICUs at CHRU-Nancy (France) in patients with severe COVID-19 during the three initial waves of COVID-19. The second aims were to analyze resistance and to describe the antibiotic treatments. We conducted a retrospective cohort study among adult patients who were hospitalized for acute respiratory distress syndrome due to COVID-19 and who developed a hospital-acquired infection caused by during their ICU stay. Among the 51 patients included, most were male (90%) with comorbidities (77%), and the first identification of infection occurred after a median ICU stay of 11 days. Several patients acquired infections with MDR (27%) and XDR (8%) strains. The agents that strains most commonly exhibited resistance to were penicillin + β-lactamase inhibitors (59%), cephalosporins (42%), monobactams (32%), and carbapenems (27%). Probabilistic antibiotic treatment was prescribed for 49 patients (96%) and was subsequently adapted for 51% of patients after antibiogram and for 33% of patients after noncompliant antibiotic plasma concentration. Hospital-acquired infection is a common and life-threatening complication in critically ill patients. Efforts to minimize the occurrence and improve the treatment of such infections, including infections caused by resistant strains, must be pursued.
PubMed: 38786119
DOI: 10.3390/antibiotics13050390 -
BMC Microbiology May 2024The emergence of multidrug-resistant (MDR) Escherichia coli strains poses significant challenges in clinical settings, particularly when these strains harbor New Delhi...
Unveiling the genetic architecture and transmission dynamics of a novel multidrug-resistant plasmid harboring bla in E. Coli ST167: implications for antibiotic resistance management.
BACKGROUND
The emergence of multidrug-resistant (MDR) Escherichia coli strains poses significant challenges in clinical settings, particularly when these strains harbor New Delhi metallo-ß-lactamase (NDM) gene, which confer resistance to carbapenems, a critical class of last-resort antibiotics. This study investigates the genetic characteristics and implications of a novel bla-carrying plasmid pNDM-5-0083 isolated from an E. coli strain GZ04-0083 from clinical specimen in Zhongshan, China.
RESULTS
Phenotypic and genotypic evaluations confirmed that the E. coli ST167 strain GZ04-0083 is a multidrug-resistant organism, showing resistance to diverse classes of antibiotics including ß-lactams, carbapenems, fluoroquinolones, aminoglycosides, and sulfonamides, while maintaining susceptibility to monobactams. Investigations involving S1 pulsed-field gel electrophoresis, Southern blot analysis, and conjugation experiments, alongside genomic sequencing, confirmed the presence of the bla gene within a 146-kb IncFIB plasmid pNDM-5-0083. This evidence underscores a significant risk for the horizontal transfer of resistance genes among bacterial populations. Detailed annotations of genetic elements-such as resistance genes, transposons, and insertion sequences-and comparative BLAST analyses with other bla-carrying plasmids, revealed a unique architectural configuration in the pNDM-5-0083. The MDR region of this plasmid shares a conserved gene arrangement (repA-IS15DIV-bla-ble-IS91-suI2-aadA2-dfrA12) with three previously reported plasmids, indicating a potential for dynamic genetic recombination and evolution within the MDR region. Additionally, the integration of virulence factors, including the iro and sit gene clusters and enolase, into its genetic architecture poses further therapeutic challenges by enhancing the strain's pathogenicity through improved host tissue colonization, immune evasion, and increased infection severity.
CONCLUSIONS
The detailed identification and characterization of pNDM-5-0083 enhance our understanding of the mechanisms facilitating the spread of carbapenem resistance. This study illuminates the intricate interplay among various genetic elements within the novel bla-carrying plasmid, which are crucial for the stability and mobility of resistance genes across bacterial populations. These insights highlight the urgent need for ongoing surveillance and the development of effective strategies to curb the proliferation of antibiotic resistance.
Topics: Plasmids; Escherichia coli; Drug Resistance, Multiple, Bacterial; Anti-Bacterial Agents; beta-Lactamases; Humans; Escherichia coli Infections; Microbial Sensitivity Tests; China; Gene Transfer, Horizontal; Carbapenems
PubMed: 38783210
DOI: 10.1186/s12866-024-03333-1 -
Phytopathology May 2024The breeding of disease-resistant soybeans cultivars to manage Phytophthora root and stem rot caused by the pathogen involves combining quantitative disease resistance...
The breeding of disease-resistant soybeans cultivars to manage Phytophthora root and stem rot caused by the pathogen involves combining quantitative disease resistance (QDR) and gene-mediated resistance. To identify and confirm potential mechanisms of QDR towards , we conducted a time course study comparing changes in gene expression among Conrad and M92-220 with high QDR to susceptible genotypes, Sloan and 3 mutants derived from fast neutron (FN) irradiation of M92-220. Differentially expressed genes from Conrad and M92-220 indicated several shared defense-related pathways at the transcriptomic level, but also defense pathways unique to each cultivar such as stilbenoid, diarylheptanoid and gingerol biosynthesis, and monobactam biosynthesis. Gene Ontology pathway analysis showed that the susceptible FN mutants lacked enrichment of three terpenoid related-pathways and two cell wall-related pathways at either one or both timepoints, in contrast to M92-220. The susceptible mutants also lacked enrichment of potentially important KEGG pathways at either one or both timepoints, including sesquiterpenoid and triterpenoid biosynthesis, thiamine metabolism, arachidonic acid, stilbenoid, diarylheptanoid and gingerol biosynthesis, and monobactam biosynthesis. Additionally, thirty-one genes which were differentially expressed in M92-220 following infection were not expressed in the mutants. These 31 genes have annotations related to unknown proteins, valine, leucine, and isoleucine biosynthesis and protein and lipid metabolic processes. The results of this study confirm previously proposed mechanisms of QDR, provide evidence for potential novel QDR pathways in M92-220, and furthers our understanding of the complex network associated with QDR mechanisms in soybean towards .
PubMed: 38772042
DOI: 10.1094/PHYTO-11-23-0436-R -
PloS One 2024NDM-producing carbapenem-resistant bacterial infections became a challenge for clinicians. Combination therapy of aztreonam and ceftazidime-avibactam is a prudent choice...
NDM-producing carbapenem-resistant bacterial infections became a challenge for clinicians. Combination therapy of aztreonam and ceftazidime-avibactam is a prudent choice for these infections. However, there is still no recommendation of a practically feasible method for testing aztreonam and ceftazidime-avibactam synergy. We proposed a simple method for testing aztreonam and ceftazidime-avibactam synergy and compared it with reference broth micro-dilution and other methods. Carbapenem-resistant Enterobacterales clinical isolates were screened for the presence of the NDM gene by the Carba R test. NDM harbouring isolates were tested for aztreonam and ceftazidime-avibactam synergy by broth microdilution (reference method), E strip-disc diffusion, double disc diffusion, and disc replacement methods. In the newly proposed method, the MHA medium was supplemented with ceftazidime-avibactam (corresponding to an aztreonam concentration of 4μg/ml). The MHA medium was then inoculated with the standard inoculum (0.5 McFarland) of the test organism. An AZT disc (30 μg) was placed on the supplemented MHA medium, and the medium was incubated overnight at 37°C. Aztreonam zone diameter on the supplemented MHA medium (in the presence of ceftazidime-avibactam) was compared with that from a standard disc diffusion plate (without ceftazidime-avibactam), performed in parallel. Interpretation of synergy was based on the restoration of aztreonam zone diameter (in the presence of ceftazidime-avibactam) crossing the CLSI susceptibility breakpoint, i.e., ≥ 21 mm. Of 37 carbapenem-resistant NDM-producing isolates, 35 (94.6%) were resistant to aztreonam and tested synergy positive by the proposed method. Its sensitivity and specificity were 97.14% and 100%, respectively. Cohen's kappa value showed substantial agreement of the reference method with the proposed method (κ = 0.78) but no other methods. The proposed method is simple, easily interpretable, and showed excellent sensitivity, specificity, and agreement with the reference method. Therefore, the new method is feasible and reliable for testing aztreonam synergy with avibactam in NDM-producing Enterobacterales.
Topics: Ceftazidime; Aztreonam; Azabicyclo Compounds; Drug Combinations; beta-Lactamases; Microbial Sensitivity Tests; Anti-Bacterial Agents; Enterobacteriaceae; Humans; Drug Synergism; Enterobacteriaceae Infections
PubMed: 38758757
DOI: 10.1371/journal.pone.0303753 -
Microbiology Spectrum Jun 2024This study aimed to assess the efficacy of ceftazidime-avibactam (CZA) in combination with various antimicrobial agents against carbapenem-resistant (CRKP). We...
UNLABELLED
This study aimed to assess the efficacy of ceftazidime-avibactam (CZA) in combination with various antimicrobial agents against carbapenem-resistant (CRKP). We selected 59 clinical CRKP isolates containing distinct drug resistance mechanisms. The minimum inhibitory concentrations (MICs) of meropenem (MEM), colistin (COL), eravacycline (ERA), amikacin (AK), fosfomycin (FOS), and aztreonam (ATM), both individually and in combination with CZA, were tested using the checkerboard method. The interactions of antimicrobial agent combinations were assessed by fractional inhibitory concentration index (FICI) and susceptible breakpoint index (SBPI). The time-kill curve assay was employed to dynamically evaluate the effects of these drugs alone and in combination format. In the checkerboard assay, the combination of CZA+MEM showed the highest level of synergistic effect against both KPC-producing and carbapenemase-non-producing isolates, with synergy rates of 91.3% and 100%, respectively. Following closely was the combination of FOS+CZA . For metallo-beta-lactamases (MBLs) producing strains, ATM+CZA displayed complete synergy, while the combination of MEM+CZA showed a synergy rate of only 57.14% for NDM-producing strains and 91.67% for IMP-producing strains. In the time-kill assay, MEM+CZA also demonstrated significant synergistic effects against the two KPC-2-producing isolates (Y070 and L70), the two carbapenemase-non-producing isolates (Y083 and L093), and the NDM-1-producing strain L13, with reductions in log CFU/mL exceeding 10 compared to the control. Against the IMP-producing strain Y047, ATM+CZA exhibited the highest synergistic effect, resulting in a log CFU/mL reduction of 10.43 compared to the control. The combination of CZA and MEM exhibited good synergistic effects against KPC-producing and non-enzyme-producing strains, followed by the FOS+CZA combination. Among MBL-producing strains, ATM+CZA demonstrated the most pronounced synergistic effect. However, the combinations of CZA with ERA, AK, and COL show irrelevant effects against the tested clinical isolates.
IMPORTANCE
Our study confirmed the efficacy of the combination CZA+MEM against KPC-producing and non-carbapenemase-producing strains. For metalloenzyme-producing strains, CZA+ATM demonstrated the most significant synergy. Additionally, CZA exhibited a notable synergy effect when combined with FOS. These combination therapies present promising new options for the treatment of CRKP infection.
Topics: Azabicyclo Compounds; Klebsiella pneumoniae; Ceftazidime; Drug Combinations; Microbial Sensitivity Tests; Humans; Anti-Bacterial Agents; Klebsiella Infections; Carbapenem-Resistant Enterobacteriaceae; Drug Synergism; beta-Lactamases; Carbapenems; Drug Resistance, Multiple, Bacterial; Bacterial Proteins; Fosfomycin; Aztreonam
PubMed: 38712934
DOI: 10.1128/spectrum.00107-24 -
Microbial Pathogenesis Jul 2024The emergence of carbapenem-resistant Pseudomonas putida (CRPP) has raised public awareness. This study investigated two strains from the Pseudomonas putida group that...
OBJECTIVES
The emergence of carbapenem-resistant Pseudomonas putida (CRPP) has raised public awareness. This study investigated two strains from the Pseudomonas putida group that were resistant to carbapenem, tigecycline, and aztreonam-avibactam (ATM-AVI), with a focus on their microbial and genomic characteristics.
METHODS
We assessed the antibiotic resistance profile using broth dilution, disk diffusion, and E-test methods. Efflux pump phenotype testing and real-time quantitative PCR were employed to evaluate efflux pump activity in tigecycline resistance, while polymerase chain reaction was utilized to detect common carbapenem genes. Additionally, whole-genome sequencing was performed to analyze genomic characteristics. The transferability of bla and bla was assessed through a conjugation experiment. Furthermore, growth kinetics and biofilm formation were examined using growth curves and crystal violet staining.
RESULTS
Both strains demonstrated resistance to carbapenem, tigecycline, and ATM-AVI. Notably, NMP can restore sensitivity to tigecycline. Subsequent analysis revealed that they co-produced bla, bla, tmexCD-toprJ, and bla, belonging to a novel sequence type ST268. Although they were closely related on the phylogenetic tree, they exhibited different levels of virulence. Genetic environment analysis indicated variations compared to prior studies, particularly regarding the bla and bla genes, which showed limited horizontal transferability. Moreover, it was observed that temperature exerted a specific influence on their biological factors.
CONCLUSION
We initially identified two P. putida ST268 strains co-producing bla, bla, bla, and tmexCD-toprJ. The resistance to tigecycline and ATM-AVI can be attributed to the presence of multiple drug resistance determinants. These findings underscore the significance of P. putida as a reservoir for novel antibiotic resistance genes. Therefore, it is imperative to develop alternative antibiotic therapies and establish effective monitoring of bacterial resistance.
Topics: Pseudomonas putida; Tigecycline; Anti-Bacterial Agents; Microbial Sensitivity Tests; China; Aztreonam; Azabicyclo Compounds; beta-Lactamases; Drug Resistance, Multiple, Bacterial; Whole Genome Sequencing; Humans; Drug Combinations; Biofilms; Bacterial Proteins; Pseudomonas Infections; Carbapenems
PubMed: 38697232
DOI: 10.1016/j.micpath.2024.106668 -
The Science of the Total Environment Jun 2024Carbapenem resistance's global proliferation poses a significant public health challenge. The primary resistance mechanism is carbapenemase production. In this study, we...
Carbapenem resistance's global proliferation poses a significant public health challenge. The primary resistance mechanism is carbapenemase production. In this study, we discovered a novel carbapenemase, RATA, located on the chromosome of Riemerella anatipestifer isolates. This enzyme shares ≤52 % amino acid sequence identity with other known β-lactamases. Antimicrobial susceptibility tests and kinetic assays demonstrated that RATA could hydrolyze not only penicillins and extended-spectrum cephalosporins but also monobactams, cephamycins, and carbapenems. Furthermore, its activity was readily inhibited by β-lactamase inhibitors. Bioinformatic analysis revealed 46 bla-like genes encoding 27 variants in the NCBI database, involving 21 different species, including pathogens, host-associated bacteria, and environmental isolates. Notably, bla-positive strains were globally distributed and primarily collected from marine environments. Concurrently, taxonomic analysis and GC content analysis indicated that bla orthologue genes were predominantly located on the chromosomes of Flavobacteriaceae and shared a similar GC content as Flavobacteriaceae. Although no explicit mobile genetic elements were identified by genetic environment analysis, bla possessed the ability of horizontal transfer in R. anatipestifer via natural transformation. This work's data suggest that RATA is a new chromosome-encoded class A carbapenemase, and Flavobacteriaceae from marine environments could be the primary reservoir of the bla gene.
Topics: beta-Lactamases; Bacterial Proteins; Anti-Bacterial Agents; Microbial Sensitivity Tests; Carbapenems
PubMed: 38692330
DOI: 10.1016/j.scitotenv.2024.172873