Recombinase-aided amplification assay for rapid detection of imipenem-resistant Pseudomonas aeruginosa and rifampin-resistant Pseudomonas aeruginosa.

The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr, in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr, the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/µL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.

The OprF porin as a potential target for the restoration of carbapenem susceptibility in Pseudomonas aeruginosa expressing acquired carbapenemases.

Carbapenem resistance in Pseudomonas aeruginosa is primarily due to the acquisition of carbapenemases and is often associated with a diminution of the membrane permeability. The outer membrane protein, OprD, is a well-known route, by which carbapenems, predominantly imipenem, can enter the cell, and its loss has been associated with reduced susceptibility to imipenem. In this study, we investigated the antibiotic susceptibility patterns of isogenic P. aeruginosa mutants containing various acquired ß-lactamases, including carbapenemases, in a porin-depleted background. We identified that the deletion of oprF was associated with some recovery of susceptibility to carbapenems.

Association of OPRD1 Gene Variants with Changes in Body Weight and Psychometric Indicators in Patients with Eating Disorders.

Objectives: This study aimed to investigate whether genetic variations in the OPRD1 gene affect psychopathological symptoms and personality dimensions in eating disorders (ED) patients and/or contribute to ED risk. Methods: The study involved 221 female patients with anorexia nervosa (AN), 88 with bulimia nervosa (BN), and 396 controls. Sixteen tag-single nucleotide polymorphisms (SNPs) in OPRD1 were identified. Psychometric evaluations were conducted using the Symptom Checklist 90 Revised (SCL-90R) and the Eating Disorders Inventory Test-2 (EDI-2). p-values obtained by regression models were corrected for multiple testing by the False Discovery Rate (FDR) method. Results: In AN patients, genotypes rs204077TT and rs169450TT were linked to lower body-mass index (BMI) values (FDR-q = 0.035 and 0.017, respectively), as was rs2234918 in a log-additive model (BMI: 18.0 ± 0.28, 17.22 ± 0.18 and 16.59 ± 0.39 for TT, TC and CC carriers, FDR-q = 0.012). Additionally, AN patients carrying the rs72665504AA genotype had higher scores in interpersonal distrust (FDR-q = 0.030), whilst BN carriers of rs513269TT and rs2873795TT showed lower scores in ineffectiveness (FDR-q = 0.041 and FDR-q = 0.021). In the AN group, BMI correlated with variability in a distal haplotype (rs508448/rs204077/rs223491, FDR-q = 0.028), which was also associated with the global positive symptom total (PST) index of SCL-90R (FDR-q = 0.048). Associations were more noticeable in BN patients; again, the distal region of the gene was linked to EDI-2 total scores (FDR-q = 0.004-0.048 for the four last haplotypes) and two global SCL-90R indices (GSI: FDR-q = 0.011 and positive symptom distress index (PSDI): FDR-q = 0.003 for the last s204077/rs2234918/rs169450 combination). No associations with ED risk were observed. Conclusions: Genetic variation in the OPRD1 gene, particularly in its distal region, is associated with BMI and psychopathological comorbidities in ED patients.

The Frequency of AmpC Overproduction, OprD Downregulation and OprM Efflux Pump Expression in Pseudomonas aeruginosa: A Comprehensive Meta-Analysis.

BACKGROUND: Pseudomonas aeruginosa is a major opportunistic pathogen responsible for a wide range of infections. The emergence of antibiotic resistance in this pathogen poses a significant public health challenge. This study aims to conduct a comprehensive meta-analysis of studies conducted in Iran to determine the frequency of key antibiotic resistance mechanisms in Pseudomonas aeruginosa and their association with multidrug-resistant and extensively drug-resistant strains or pandrug-resistant strains. METHODS: Systematic database searches encompassing literature up to June 2023 were undertaken. The selected studies centered on OprD downregulation, efflux pump (mexAB-OprM, mexXY-OprM) expression, and AmpC overproduction. Extracted data were synthesized in a meta-analysis for pooled frequency determination of each resistance mechanism. RESULTS: In total, 24 studies were included. OprD downregulation exhibited a pooled frequency of 61%. Efflux pump component frequency ranged from 48% to 77.5%. AmpC overproduction was identified in 29.1% of isolates. Polymyxin B and colistin demonstrated lower antibiotic resistance rates, with pooled frequency of 1% and 1.6%, respectively. Conversely, resistance to other antibiotics ranged widely, with pooled frequency spanning 38.4% to 98.2%. CONCLUSION: This study underscores the concerning frequency of diverse antibiotic resistance mechanisms in Pseudomonas aeruginosa strains from Iran. Concurrent OprD downregulation, mexAB, mexXY, OprM expression, and AmpC overproduction highlight the urgent need for stringent infection control and prudent antibiotic usage to curb the dissemination of these resistant strains. PROSPERO: CRD42022379311.

AmpC induction by imipenem in Pseudomonas aeruginosa occurs in the absence of OprD and impacts imipenem/relebactam susceptibility.

In the United States, carbapenem resistance in Pseudomonas aeruginosa is linked to the regulation of chromosomal resistance determinants, AmpC and OprD. The ß-lactamase AmpC requires overexpression and genetic modifications to be capable of inhibiting imipenem activity. The outer membrane porin OprD can be downregulated or undergo genetic modifications that strongly correlate with imipenem non-susceptibility. Co-administration of imipenem and the ß-lactamase inhibitor, relebactam, can lower imipenem minimal inhibitory concentrations and restore susceptibility. However, it is not understood how this occurs in P. aeruginosa isolates that do not overproduce AmpC or produce a functional OprD for imipenem entry. Therefore, we investigated whether imipenem could enter P. aeruginosa in the absence of OprD and whether any of the chromosomal ß-lactamases (AmpC, OXA-50, and PIB-1) contributed to imipenem and/or imipenem/relebactam non-susceptibility. This investigation evaluated 17 imipenem non-susceptible clinical isolates and three laboratory strains of PAO1, two of which were porin transposon mutants for either oprD or opdP. Expression of OXA-50 and PIB-1 RNA was similar to PAO1. However, all 20 isolates exhibited blaampC induction under sublethal exposure to imipenem. This occurred in the absence of detectable OprD protein in 18 isolates. Collectively, our data identify that (i) OprD was not the only channel required for imipenem entry and (ii) imipenem susceptibility can be restored by imipenem/relebactam due to the interaction between relebactam and blaampC overexpression due to imipenem induction.IMPORTANCEInfections caused by Pseudomonas aeruginosa are associated with high mortality and worsened clinical outcomes. The carbapenem, imipenem, has been combined with the ß-lactamase inhibitor relebactam to treat carbapenem-resistant P. aeruginosa. Downregulation of the outer membrane porin, OprD is the major mechanism of imipenem resistance; however, relebactam inhibits the chromosomally encoded AmpC ß-lactamase. We investigated how relebactam was able to reduce P. aeruginosa imipenem minimal inhibitory concentrations (MICs) in isolates in which OprD was downregulated. Our data show that imipenem is capable of entering the cell in the absence of OprD and capable of inducing the AmpC ß-lactamase. The induction of AmpC provides a substrate for relebactam, impacting the imipenem MIC. The data presented support the use of an alternative porin(s) for entry of imipenem. This study provides the basis for further investigation into modifications of imipenem or similar molecules that would increase the affinity for other porins in isolates resistant to imipenem.

In-silico screening of missense nsSNPs in Delta-opioid receptor protein and their restoring tendency on MCRT interaction; focusing on dynamic nature.

Delta-opioid receptor protein (OPRD1) is one of the potential targets for treating pain. The presently available opioid agonists are known to cause unnecessary side effects. To discover a novel opioid agonist, our research group has synthesized a chimeric peptide MCRT and proved its potential activity through in vivo analysis. Non-synonymous SNPs (nsSNPs) missense mutations affect the functionality and stability of proteins leading to diseases. The current research was focused on understanding the role of MCRT in restoring the binding tendency of OPRD1 nsSNPs missense mutations on dynamic nature in comparison with Deltorphin-II and morphiceptin. The deleterious effects of nsSNPs were analyzed using various bioinformatics tools for predicting structural, functional, and oncogenic influence. The shortlisted nine nsSNPs were predicted for allergic reactions, domain changes, post-translation modification, multiple sequence alignment, secondary structure, molecular dynamic simulation (MDS), and peptide docking influence. Further, the docked complex of three shortlisted deleterious nsSNPs was analyzed using an MDS study, and the highly deleterious shortlisted nsSNP A149T was further analyzed for higher trajectory analysis. MCRT restored the binding tendency influence caused by nsSNPs on the dynamics of stability, functionality, binding affinity, secondary structure, residues connection, motion, and folding of OPRD1 protein.

Whole-genome sequencing, multilocus sequence typing, and resistance mechanism of the carbapenem-resistant Pseudomonas aeruginosa in China.

Pseudomonas aeruginosa is a significant pathogen responsible for severe multisite infections with high morbidity and mortality rates. This study analyzed carbapenem-resistant Pseudomonas aeruginosa (CRPA) at a tertiary hospital in Shandong, China, using whole-genome sequencing (WGS). The objective was to explore the mechanisms and molecular characteristics of carbapenem resistance. A retrospective analysis of 91 isolates from January 2022 to March 2023 was performed, which included strain identification and antimicrobial susceptibility testing. WGS was utilized to determine the genome sequences of these CRPA strains, and the species were precisely identified using average nucleotide identification (ANI), with further analysis on multilocus sequence typing and strain relatedness. Some strains were found to carry the ampD and oprD genes, while only a few harbored carbapenemase genes or related genes. Notably, all strains possessed the mexA, mexE, and mexX genes. The major lineage identified was ST244, followed by ST235. The study revealed a diverse array of carbapenem resistance mechanisms among hospital isolates, differing from previous studies in mainland China. It highlighted that carbapenem resistance is not due to a single mechanism but rather a combination of enzyme-mediated resistance, AmpC overexpression, OprD dysfunction, and efflux pump overexpression. This research provides valuable insights into the evolutionary mechanisms and molecular features of CRPA resistance in this region, aiding in the national prevention and control of CRPA, and offering references for targeting and developing new drugs.

In Silico Molecular Analysis of Carbapenemase-Negative Carbapenem-Resistant Pseudomonas aeruginosa Strains in Greece.

To date, three carbapenem resistance mechanisms have been identified: carbapenemase released from the pathogen, changes in the expression of the outer membrane OprD porin, and overexpression of the efflux pump MexAB-OprM. Twelve carbapenemase-negative carbapenem-resistant Pseudomonas aeruginosa strains, isolated from patients hospitalized at the University Hospital of Larissa, Central Greece, during 2023, which belonged to various sequence types (STs), were selected and were studied focusing on the characterization of their ß-lactamases, on changes to OprD and its regulator MexT proteins, and on alterations to the MexAB-OprM regulator proteins encoded by the mexR, nalC, and nalD genes. Whole genome sequencing analysis revealed the presence of ß-lactamase encoding genes, with blaPAO present in all isolates. Additionally, seven different genes of the oxacillinase family (blaOXA-35, blaOXA-50, blaOXA-395, blaOXA-396, blaOXA-486, blaOXA-488, blaOXA-494) were identified, with each strain harboring one to three of these. Regarding the OprD, five strains had truncated structures, at Loop 2, Loop 3, Loop 4, and Loop 9, while the remaining strains carried previously reported amino acid changes. Further, an additional strain had a truncated MexR; whereas, two other strains had totally modified NalC sequences. The active form of MexT, responsible for the downregulation of OprD production, as the intact sequence of the NalD protein, was found in all the strains studied. It is concluded that the truncated OprD, MexR, and NalC proteins, detected in eight strains, probably led to inactive proteins, contributing to carbapenem resistance. However, four strains carried known modifications in OprD, MexR, and NalC, as previously reported in both susceptible and resistant strains, a finding that indicates the complexity of carbapenem resistance in P. aeruginosa.

Clonal Distribution and Its Association With the Carbapenem Resistance Mechanisms of Carbapenem-Non-Susceptible Pseudomonas aeruginosa Isolates From Korean Hospitals.

Background: Carbapenem resistance in Pseudomonas aeruginosa is a serious global health problem. We investigated the clonal distribution and its association with the carbapenem resistance mechanisms of carbapenem-non-susceptible P. aeruginosa isolates from three Korean hospitals. Methods: A total of 155 carbapenem-non-susceptible P. aeruginosa isolates collected between 2011 and 2019 were analyzed for sequence types (STs), antimicrobial susceptibility, and carbapenem resistance mechanisms, including carbapenemase production, the presence of resistance genes, OprD mutations, and the hyperproduction of AmpC ß-lactamase. Results: Sixty STs were identified in carbapenem-non-susceptible P. aeruginosa isolates. Two high-risk clones, ST235 (N=41) and ST111 (N=20), were predominant; however, sporadic STs were more prevalent than high-risk clones. The resistance rate to amikacin was the lowest (49.7%), whereas that to piperacillin was the highest (92.3%). Of the 155 carbapenem-non-susceptible isolates, 43 (27.7%) produced carbapenemases. Three metallo-ß-lactamase (MBL) genes, blaIMP-6 (N=38), blaVIM-2 (N=3), and blaNDM-1 (N=2), were detected. blaIMP-6 was detected in clonal complex 235 isolates. Two ST773 isolates carried blaNDM-1 and rmtB. Frameshift mutations in oprD were identified in all isolates tested, regardless of the presence of MBL genes. Hyperproduction of AmpC was detected in MBL gene-negative isolates. Conclusions: Frameshift mutations in oprD combined with MBL production or hyperproduction of AmpC are responsible for carbapenem resistance in P. aeruginosa. Further attention is required to curb the emergence and spread of new carbapenem-resistant P. aeruginosa clones.

Regulation of headache response and transcriptomic network by the trigeminal ganglion clock.

OBJECTIVE: To characterize the circadian features of the trigeminal ganglion in a mouse model of headache. BACKGROUND: Several headache disorders, such as migraine and cluster headache, are known to exhibit distinct circadian rhythms of attacks. The circadian basis for these rhythmic pain responses, however, remains poorly understood. METHODS: We examined trigeminal ganglion ex vivo and single-cell cultures from Per2::LucSV reporter mice and performed immunohistochemistry. Circadian behavior and transcriptomics were investigated using a novel combination of trigeminovascular and circadian models: a nitroglycerin mouse headache model with mechanical thresholds measured every 6 h, and trigeminal ganglion RNA sequencing measured every 4 h for 24 h. Finally, we performed pharmacogenomic analysis of gene targets for migraine, cluster headache, and trigeminal neuralgia treatments as well as trigeminal ganglion neuropeptides; this information was cross-referenced with our cycling genes from RNA sequencing data to identify potential targets for chronotherapy. RESULTS: The trigeminal ganglion demonstrates strong circadian rhythms in both ex vivo and single-cell cultures, with core circadian proteins found in both neuronal and non-neuronal cells. Using our novel behavioral model, we showed that nitroglycerin-treated mice display circadian rhythms of pain sensitivity which were abolished in arrhythmic Per1/2 double knockout mice. Furthermore, RNA-sequencing analysis of the trigeminal ganglion revealed 466 genes that displayed circadian oscillations in the control group, including core clock genes and clock-regulated pain neurotransmitters. In the nitroglycerin group, we observed a profound circadian reprogramming of gene expression, as 331 of circadian genes in the control group lost rhythm and another 584 genes gained rhythm. Finally, pharmacogenetics analysis identified 10 genes in our trigeminal ganglion circadian transcriptome that encode target proteins of current medications used to treat migraine, cluster headache, or trigeminal neuralgia. CONCLUSION: Our study unveiled robust circadian rhythms in the trigeminal ganglion at the behavioral, transcriptomic, and pharmacogenetic levels. These results support a fundamental role of the clock in pain pathophysiology. PLAIN LANGUAGE SUMMARY: Several headache diseases, such as migraine and cluster headache, have headaches that occur at the same time each day. We learned that the trigeminal ganglion, an important pain structure in several headache diseases, has a 24-hour cycle that might be related to this daily cycle of headaches. Our genetic analysis suggests that some medications may be more effective in treating migraine and cluster headache when taken at specific times of the day.