Category: 3. Business

Industrikraft in Sweden AB was formed in June 2024 to support the expansion of the Swedish electricity supply. Industrikraft is now entering the next phase of nuclear power investment with the following companies – ABB, Alfa Laval, Boliden, Hitachi Energy, Höganäs AB, SSAB, Saab, Stora Enso, and the Volvo Group.

Vattenfall and Industrikraft have now signed an agreement that allows the parties to progress in enabling new nuclear power on the Värö Peninsula at Ringhals. The agreement covers various areas such as responsibilities, influence, and financing.

The next step in the collaboration is for Industrikraft to become a 20 percent shareholder in Videberg Kraft AB. The government has previously announced that the state also intends to become a shareholder in the company.

“Industrikraft and Vattenfall have reached consensus on the most important issues”, says Tom Erixon, chairman of Industrikraft and CEO of Alfa Laval. In a first step, Industrikraft will invest SEK 400 million in the project. In addition to co-financing the project company, the industry contributes with resources, expertise in project management and selection of technologies. Swedish technology has a place in a competitive environment to drive the development of a strong supplier cluster in Europe.

“It is very positivenews that Vattenfall and Industrikraft are now taking the next step to move the project forward together. The industry is an important partner in enabling new nuclear power in Sweden. The commitment and willingness of these companies to co-own the project on the Värö Peninsula is a sign that there will be a demand for the new fossil-free electricity production capacity. In the next step, Videberg Kraft AB is working to prepare an application for state risk-sharing”, says Anna Borg, CEO of Vattenfall.

The board of Pine Cliff Energy Ltd. (TSE:PNE) has announced that it will pay a dividend on the 28th of November, with investors receiving CA$0.0013 per share. Including this payment, the dividend yield on the stock will be 1.8%, which is a modest boost for shareholders’ returns.

While the dividend yield is important for income investors, it is also important to consider any large share price moves, as this will generally outweigh any gains from distributions. Investors will be pleased to see that Pine Cliff Energy’s stock price has increased by 35% in the last 3 months, which is good for shareholders and can also explain a decrease in the dividend yield.

AI is about to change healthcare. These 20 stocks are working on everything from early diagnostics to drug discovery. The best part – they are all under $10bn in marketcap – there is still time to get in early.

Even a low dividend yield can be attractive if it is sustained for years on end. Pine Cliff Energy is not generating a profit, but its free cash flows easily cover the dividend, leaving plenty for reinvestment in the business. We generally think that cash flow is more important than accounting measures of profit, so we are fairly comfortable with the dividend at this level.

Over the next year, EPS might fall by 9.6% based on recent performance. This means that the company won’t turn a profit over the next year, but with healthy cash flows at the moment the dividend could still be okay to continue.

Check out our latest analysis for Pine Cliff Energy

Looking back, the dividend has been unstable but with a relatively short history, we think it may be a bit early to draw conclusions about long term dividend sustainability. The annual payment during the last 4 years was CA$0.0996 in 2021, and the most recent fiscal year payment was CA$0.015. The dividend has fallen 85% over that period. Declining dividends isn’t generally what we look for as they can indicate that the company is running into some challenges.

Dividends have been going in the wrong direction, so we definitely want to see a different trend in the earnings per share. It’s not great to see that Pine Cliff Energy’s earnings per share has fallen at approximately 9.6% per year over the past five years. Declining earnings will inevitably lead to the company paying a lower dividend in line with lower profits.

Overall, we don’t think this company makes a great dividend stock, even though the dividend wasn’t cut this year. In the past, the payments have been unstable, but over the short term the dividend could be reliable, with the company generating enough cash to cover it. This company is not in the top tier of income providing stocks.

Market movements attest to how highly valued a consistent dividend policy is compared to one which is more unpredictable. Still, investors need to consider a host of other factors, apart from dividend payments, when analysing a company. For example, we’ve identified 2 warning signs for Pine Cliff Energy (1 is a bit concerning!) that you should be aware of before investing. If you are a dividend investor, you might also want to look at our curated list of high yield dividend stocks.

Have feedback on this article? Concerned about the content? Get in touch with us directly. Alternatively, email editorial-team (at) simplywallst.com.

This article by Simply Wall St is general in nature. We provide commentary based on historical data and analyst forecasts only using an unbiased methodology and our articles are not intended to be financial advice. It does not constitute a recommendation to buy or sell any stock, and does not take account of your objectives, or your financial situation. We aim to bring you long-term focused analysis driven by fundamental data. Note that our analysis may not factor in the latest price-sensitive company announcements or qualitative material. Simply Wall St has no position in any stocks mentioned.

Introduction

Heart failure (HF) is a primary manifestation of the end stage of various cardiovascular diseases, characterized by reduced cardiac pumping function that fails to meet the body’s metabolic demands. This condition is associated with high morbidity, hospitalization rates, and mortality, significantly impairing patients’ quality of life and imposing a substantial burden on social, economic, and healthcare resources.^1,2 Statistics indicate that approximately 64 million people worldwide suffer from heart failure, with its prevalence increasing significantly with age, particularly among the elderly population.^3–5 Despite continuous advancements in modern medical technology, the treatment of heart failure remains a formidable challenge. Effectively improving cardiac function, reducing rehospitalization rates, and lowering mortality are critical issues in clinical research.

In recent years, sacubitril/valsartan, a novel angiotensin receptor-neprilysin inhibitor (ARNI), has emerged as a promising therapeutic option for heart failure. Numerous clinical studies have demonstrated that sacubitril/valsartan not only significantly improves cardiac function indicators but also reduces hospitalization rates and all-cause mortality in heart failure patients.^6–8 This study aims to retrospectively analyze the effect of conventional cardiology drug therapy combined with sacubitril/valsartan on improving cardiac function in heart failure patients. It further evaluates its efficacy in regulating neuroendocrine function, reversing ventricular remodeling, and enhancing patients’ quality of life and exercise tolerance. The findings will provide more clinical evidence for comprehensive heart failure treatment while validating the safety and efficacy of sacubitril/valsartan, offering guidance for optimizing therapeutic strategies.

Subjects and Methods

Study Subjects

This study included patients diagnosed and treated for heart failure at our hospital between March 2022 and December 2023 to ensure a representative sample. Following a rigorous and comprehensive screening based on inclusion criteria, a total of 110 patients were enrolled. Patients were divided into a control group and an experimental group based on treatment methods. The control group received conventional cardiology drug therapy, while the experimental group received conventional cardiology drug therapy combined with sacubitril/valsartan. This study has used propensity score matching (PSM) method to optimize inter group comparability, and the specific implementation is as follows: Selection of matching variables: based on clinical relevance and literature support, age, gender, NYHA cardiac function classification, course of disease, prevalence of hypertension/diabetes, baseline LVEF value, NT proBNP level, serum creatinine and eGFR were selected as matching variables to ensure coverage of key confounding factors affecting treatment selection and prognosis. Propensity score calculation: The probability of each patient receiving the combination therapy of sacubitril/valsartan was calculated using a logistic regression model. The model included the matching variables mentioned above and validated the model discrimination (C-index=0.82). Matching implementation: The 1:1 nearest neighbor matching method was used, with a caliper value set at 0.2 times the standard deviation. Finally, 55 pairs of samples were successfully matched, and the standardized differences after matching were all<0.1, confirming a significant improvement in inter group balance. Balance validation: Baseline data was compared between the matched samples, and the results showed no statistically significant differences in key indicators such as age, gender, cardiac function grading, and glomerular filtration rate (eGFR) between the two groups (P>0.05). Clinical data were collected and summarized through medical records and other methods, and statistical methods were employed for quantitative analysis of the results. This study adhered to the principles of the Declaration of Helsinki and was reviewed and approved by the Ethics Committee of The First People’s Hospital of Yongkang City, Zhejiang Province. Due to the retrospective nature of the study, our ethics committee waived the requirement for informed consent (This study only used existing medical record data and did not intervene in any patients). Privacy safeguards for all case data included: anonymization prior to data collection, with removal of personally identifiable information (eg, names, ID numbers); establishment of a tiered access control system, granting authorized researchers exclusive access to complete datasets; and presentation of aggregated clinical data in publications to eliminate individual information disclosure risks.

Sample size calculation basis and method:

This study adopts a retrospective queue design and estimates the sample size based on the following parameters:

Effect size: According to prior research, the expected difference between the experimental group and the control group in the primary endpoint (such as LVEF improvement) is 5%, with a standard deviation of 8%.

Statistical efficacy (1- β): Set at 90%, ensuring a 90% probability of detecting the true effect.

Significance level (α): 0.05 (bilateral).

Loss of follow-up rate: Reserve 10% of the sample size to compensate for missing or lost data.

Calculation formula (based on t-test):

n = (Z1−α/2+Z1−β)2×σ2/δ2

among which

Z1 − α/2=1.96 (when α = 0.05),

Z1 − β=1.28 (when β = 0.10),

σ = 8 (standard deviation),

δ = 5 (effect quantity).

Calculation result:

n = (1.96+1.28)2×82/52≈42.3

Each group requires about 43 cases, and the actual effective data is 55 cases (after matching).

Statistical validity analysis verification

Verify the statistical validity of the existing sample size through G * Power software:

Input parameters: Effect size δ=5%, standard deviation σ=8%, α=0.05, sample size n=55 for each group.

Output result: Statistical power (1- β)=92.3%, indicating that with the current sample size, there is a 92.3% probability that the study will detect the expected effect, meeting the predetermined 90% power requirement.

Inclusion and Exclusion Criteria

Inclusion Criteria: Patients met the diagnostic criteria for heart failure;^9–11 had a life expectancy of at least six months with stable vital signs; were aged 18 years or older; and had complete clinical data.

Exclusion Criteria: Patients with severe hepatic or renal dysfunction; those with endocrine disorders or autoimmune system dysfunction; individuals with a history of malignant tumors; those with severe arrhythmias, malignant hypertension, cerebral hemorrhage, or cerebral infarction; and those with known allergies to the study drugs.

Methods

Control Group

Patients assigned to the control group underwent a standard course of conventional cardiology drug therapy. This regimen encompassed the administration of two primary medications: Benazepril Hydrochloride Tablets: Manufactured by Huahai Pharmaceutical Co., Ltd., located in Zhejiang, China, and bearing the National Drug Approval Number H20233426, benazepril hydrochloride is an angiotensin-converting enzyme (ACE) inhibitor. The prescribed dosage for each intake was 5 mg, and patients were instructed to take this medication twice daily, ensuring a consistent therapeutic level over a 24-hour period. This dosing schedule was maintained for a continuous duration of 8 weeks. Metoprolol Succinate Extended-Release Tablets: Produced by AstraZeneca Pharmaceuticals, with the National Drug Approval Number J20150044, metoprolol succinate is a beta-blocker known for its extended-release properties, allowing for once-daily dosing. Initially, patients were prescribed a dose of 95 mg per day. However, the dosage could be adjusted based on individual patient tolerance, which was assessed through regular monitoring of vital signs, symptoms, and any adverse reactions. This personalized approach to dosing was also carried out over an 8-week period.

Experimental Group

Patients in the experimental group received an enhanced therapeutic protocol that built upon the foundation of the control group’s treatment, with the notable addition of sacubitril/valsartan sodium: Sacubitril/Valsartan Sodium: This medication is a combination drug that merges the actions of sacubitril, a neprilysin inhibitor, and valsartan, an angiotensin II receptor blocker (ARB). It was manufactured by Novartis Farma S.p.A., previously known as Novartis Singapore Pharmaceutical Manufacturing Private Ltd., and was repackaged by Beijing Novartis Pharmaceuticals Co., Ltd. The drug carries the National Drug Approval Number HJ20170363 (previously J20190002). Dosage and Administration: The initial dosage prescribed for sacubitril/valsartan sodium was 100 mg per dose, taken twice daily. This dosing strategy was chosen to achieve a rapid and sustained therapeutic effect. Similar to the control group’s regimen, the experimental group’s treatment span was also 8 weeks. Throughout this period, patients’ responses to the medication, including any changes in blood pressure, heart rate, and overall well-being, were closely monitored to ensure both efficacy and safety. The twice-daily dosing of sacubitril/valsartan sodium was designed to maintain stable plasma concentrations of the active ingredients, thereby optimizing the drug’s beneficial effects on cardiac function and reducing the risk of adverse cardiovascular events.

Observational Indicators

Clinical Efficacy

Clinical efficacy was categorized into three levels: marked effect, effective, and ineffective. Total efficacy was calculated as the sum of marked and effective rates.

Marked Effect

Significant alleviation of clinical symptoms after treatment, with heart function classification improving by more than two grades compared to pre-treatment.

Effective

Some improvement in clinical symptoms, with heart function classification improving by 1–2 grades compared to pre-treatment.

Ineffective

No significant improvement in clinical symptoms or heart function classification.

Cardiac Function Indicators

Cardiac function indicators included left ventricular ejection fraction (LVEF), stroke volume (SV), cardiac output (CO), left ventricular end-systolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD). These were measured using echocardiography after treatment.

Myocardial Injury

Indicators included N-terminal pro-brain natriuretic peptide (NT-proBNP), homocysteine (Hcy), and cardiac troponin I (cTnI). After treatment, 4 mL of fasting venous blood was collected from patients, centrifuged at 3,000 r/min for 10 minutes, and stored frozen. A BK-600 automated biochemical analyzer and corresponding reagent kits (Production company: Saipei Biotechnology, location: Wuhan, China) were used for detection via enzyme-linked immunosorbent assay (ELISA).

Neurohormones

Neuroendocrine hormones such as norepinephrine (NE), aldosterone (ALD), and angiotensin II (AngII) were measured. Blood samples were collected post-treatment and analyzed using radioimmunoassay methods, the reagent kit is provided by BIOESN, located in Shanghai, China.

Exercise Tolerance

The 6-minute walk test (6MWT) was used to assess exercise tolerance and cardiopulmonary function by measuring the maximum walking distance patients could achieve in 6 minutes. Measurements were taken before and after treatment. The patient walks back and forth on a 30 meter long straight line and records the maximum distance traveled within 6 minutes. Before the experiment, patients need to sit quietly and rest for 10 minutes. During the experiment, patients are allowed to rest appropriately according to their own situation.

Quality of Life

Use the Minnesota Heart Failure Quality of Life Questionnaire (MLHFQ), which covers physical, emotional, and other areas, with a total score ranging from 0 to 105, with lower scores indicating better quality of life.

Adverse Reactions

All adverse reactions were recorded, including headaches, hepatic or renal dysfunction, and hyperkalemia. The incidence of adverse reactions was calculated.

Statistical Methods

Graphs in this study were processed using GraphPad Prism 8. Data organization and analysis were performed using SPSS 26.0. Measurement data were expressed as mean ± standard deviation (), and comparisons between groups were conducted using the t-test. Count data were expressed as [n (%)], and comparisons between groups were performed using the chi-square (χ²) test. Statistical significance was set at P < 0.05.

Results

Baseline Data

The experimental group included 55 patients, aged 49–88 years (78.21 ± 9.17 years), with 24 males and 21 females. Their heart function classifications were: Class II (35 cases) and Class III (20 cases), and their disease duration ranged from 4–10 years (6.87 ± 1.25 years). The control group included 55 patients, aged 49–88 years (78.57 ± 8.35 years), with 25 males and 20 females. Their heart function classifications were: Class II (37 cases) and Class III (18 cases), and their disease duration ranged from 4–10 years (7.03 ± 1.17 years). The baseline characteristics of the two groups were comparable (P > 0.05) (Table 1).

Table 1 Comparison of Baseline Data Between the Two Groups

Clinical Efficacy

The total clinical efficacy rate in the experimental group was 94.55%, significantly higher than the 78.18% observed in the control group, with a statistically significant difference (P < 0.05). See Figure 1.

Figure 1 Comparison of Total Clinical Efficacy Rates Between the Two Groups. Note: *indicates a significant difference between the two groups, P < 0.05.

Cardiac Function Indicators

After treatment, the LVEF, SV, and CO levels in the experimental group were higher than those in the control group, while the LVESD and LVEDD levels were significantly lower in the experimental group than in the control group (P < 0.05). See Table 2.

Table 2 Comparison of Cardiac Function Indicators Between the Two Groups

Myocardial Injury

After treatment, the levels of NT-proBNP, Hcy, and cTnI in the experimental group were significantly lower than those in the control group (P < 0.05). See Figure 2.

Figure 2 Comparison of Myocardial Injury Biomarker Levels Between the Two Groups. Note: *indicates a significant difference between the two groups, P < 0.05.

Neurohormones

After treatment, the levels of NE, ALD, and AngII in the experimental group were significantly lower than those in the control group (P < 0.05). See Figure 3.

Figure 3 Comparison of Neuroendocrine Hormone Levels Between the Two Groups. Note: *indicates a significant difference between the two groups, P < 0.05.

Exercise Tolerance

After treatment, the 6MWT level in the experimental group was higher than that in the control group (P < 0.05). See Figure 4.

Figure 4 Comparison of 6MWT Levels Between the Two Groups. Note: *indicates a significant difference between the two groups, P < 0.05.

Quality of Life

After treatment, the MLHFQ score in the experimental group was lower than that in the control group (P < 0.05). See Figure 5.

Figure 5 Comparison of MLHFQ Scores Between the Two Groups. Note: *indicates a significant difference between the two groups, P < 0.05.

Adverse Reactions

There was no significant difference in the occurrence of adverse reactions between the two groups. Treatment with Sacubitril/Valsartan did not result in a significant increase in adverse reactions (P > 0.05). See Table 3.

Table 3 Comparison of Adverse Reactions Between the Two Groups

Discussion

Heart failure (HF) is a complex syndrome caused by structural and functional abnormalities of the heart, primarily characterized by decreased cardiac function, reduced exercise tolerance, and impaired quality of life. Although traditional cardiology medications can alleviate symptoms to some extent, they have limitations in improving long-term outcomes of the disease.^12–15 In recent years, Sacubitril/Valsartan, a novel drug with a unique dual mechanism of action, has demonstrated significant advantages in the treatment of heart failure. Sacubitril/Valsartan inhibits neprilysin activity, thereby enhancing the natriuretic peptide system to promote diuresis, vasodilation, and antifibrotic effects. Concurrently, it blocks angiotensin II receptors, reducing the activity of the renin-angiotensin-aldosterone system (RAAS), which alleviates cardiac burden and reduces the risk of ventricular remodeling.^16,17 This study further investigated the effects of combining Sacubitril/Valsartan with conventional cardiology medications. The results showed that this combined treatment regimen significantly improved patients’ cardiac function, enhanced exercise tolerance, and improved quality of life. These findings provide a new direction and valuable reference for the clinical treatment of heart failure.

Regarding the improvement of cardiac function, the study demonstrated that patients in the experimental group showed significantly greater improvements in cardiac function indicators, including left ventricular ejection fraction (LVEF), stroke volume (SV), and cardiac output (CO), compared to the control group. Additionally, their left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were significantly reduced. Conventional treatments for chronic heart failure typically include inotropic agents, diuretics, and vasodilators, with Benazepril Hydrochloride Tablets and Metoprolol Succinate Extended-Release Tablets being commonly used medications. Benazepril Hydrochloride, an angiotensin-converting enzyme inhibitor, works by blocking the conversion of angiotensin and inhibiting kininase activity, which reduces the degradation of bradykinin. This leads to vasodilation, reduced blood pressure, improved cardiac blood flow, and restored cardiac function. Metoprolol Succinate Extended-Release Tablets, a highly selective β1-receptor blocker, reduce heart rate and myocardial contractility, thereby decreasing myocardial oxygen demand and lowering blood pressure. Furthermore, Metoprolol can inhibit ventricular remodeling, slow the progression of heart failure, and prevent sudden cardiac death. Sacubitril/Valsartan, as a novel angiotensin receptor-neprilysin inhibitor (ARNI), combines the dual effects of inhibiting neprilysin activity and blocking angiotensin receptors. It inhibits vasoconstriction, promotes vasodilation, reduces cardiac afterload, significantly improves myocardial function, lowers the risk of ventricular remodeling, decreases rehospitalization rates, and helps stabilize the disease.^18–20 Studies have shown that combining Sacubitril/Valsartan with conventional therapeutic medications not only further enhances the heart’s pumping ability but also effectively reverses ventricular remodeling and significantly improves cardiac function. This provides more comprehensive support for the treatment of chronic heart failure.

Previous studies have found that excessive activation of the neuroendocrine system is considered one of the key factors in the pathogenesis of heart failure.^21–23 Abnormal elevation of neuroendocrine hormone levels, such as the excessive release of norepinephrine (NE), aldosterone (ALD), and angiotensin II (Ang II), not only serves as a compensatory response to heart failure but also accelerates myocardial cell remodeling, fibrosis, and the deterioration of heart function. This abnormal activation exacerbates ventricular remodeling and cell apoptosis by enhancing sympathetic nervous system excitability and the activity of the renin-angiotensin-aldosterone system (RAAS), leading to further worsening of heart failure. Therefore, inhibiting neuroendocrine activity has become a core strategy in the treatment of heart failure. Sacubitril/Valsartan, through its dual mechanism of action, has shown significant effects in inhibiting the excessive activation of the neuroendocrine system. On the one hand, as an angiotensin receptor blocker (ARB), it selectively binds to and blocks the Ang II receptor, inhibiting the vasoconstriction and sodium retention induced by Ang II, thereby reducing cardiac load, lowering vascular resistance, and improving cardiac pumping ability. On the other hand, Sacubitril/Valsartan, as a neprilysin inhibitor, enhances the function of the natriuretic peptide system, promotes vasodilation, inhibits myocardial cell hypertrophy and fibrosis, and reduces the risk of ventricular remodeling. The results of this study indicate that after combined treatment, the levels of neuroendocrine hormones in patients significantly decreased, particularly NE, ALD, and Ang II, which were notably lower than those in the control group. This suggests that Sacubitril/Valsartan can effectively regulate neuroendocrine activity, thereby delaying the progression of heart failure and reducing the incidence of long-term complications, consistent with previous similar studies. A study by domestic scholars also mentioned that Sacubitril/Valsartan could reduce the generation of Ang II by inhibiting renin release, blocking its adverse effects on the heart and blood vessels, and further alleviating cardiac burden. By reducing the excessive activation of neuroendocrine hormones, this drug not only improves cardiac function and quality of life in patients but also effectively prevents further deterioration of ventricular remodeling and cardiac tissue damage.^24–26 This study confirms the important role of Sacubitril/Valsartan in the treatment of heart failure, providing solid evidence for optimizing clinical management strategies.

Myocardial injury is an important indicator for assessing the severity of heart failure. This study shows that the levels of N-terminal pro B-type natriuretic peptide (NT-proBNP), homocysteine (Hcy), and cardiac troponin I (cTnI) in the experimental group were significantly lower than those in the control group, indicating that Sacubitril/Valsartan has a significant advantage in alleviating myocardial injury. The mechanism of action primarily involves inhibiting neprilysin activity, reducing natriuretic peptide degradation, promoting water and sodium excretion, thereby alleviating water and sodium retention, stabilizing blood pressure, and reducing cardiac load, while improving heart function. In addition, Sacubitril/Valsartan optimizes the metabolic environment of myocardial cells, reduces myocardial stress responses, significantly protects myocardial function, and suppresses the secretion of myocardial injury markers such as NT-proBNP, Hcy, and cTnI.^27,28 This result further confirms the remarkable effect of combined treatment in repairing myocardial injury, improving heart function, and protecting the myocardium, providing reliable clinical evidence for the treatment of heart failure patients.

Moreover, the 6-minute walk test (6 MWT) and the Minnesota Living with Heart Failure Questionnaire (MLHFQ) are important tools for assessing exercise tolerance and quality of life in heart failure patients. This study demonstrates that the experimental group showed significantly greater improvement in both of these indicators compared to the control group. This not only reflects the overall improvement in the functional status of patients with combined treatment but also indicates that Sacubitril/Valsartan can help patients better cope with physical activity in daily life. Drug safety is an important factor that cannot be overlooked in clinical application. In this study, the incidence of adverse reactions between the two groups showed no significant difference, and the experimental group did not experience a significant increase in adverse reaction risks due to the use of Sacubitril/Valsartan. This suggests that the drug has a high level of tolerance, providing strong support for its clinical promotion.^29–31

Significance and Limitations

This study, through retrospective analysis, confirmed the significant effect of combined conventional cardiology drug treatment and Sacubitril/Valsartan in improving heart function in heart failure patients. However, this study still has some limitations, such as a limited sample size and a short follow-up period. Future research with multi-center, large sample sizes and long-term follow-up is needed to further validate the results of this study. And that future research could consider incorporating continuous monitoring and genetic parameter surveys to more comprehensively evaluate treatment efficacy.

Conclusion

Combined conventional cardiology drug treatment and Sacubitril/Valsartan show significant advantages in the treatment of heart failure patients. This approach not only effectively improves heart function but also provides comprehensive protection by regulating neuroendocrine activity, alleviating myocardial injury, and reversing ventricular remodeling. Moreover, this treatment significantly enhances exercise tolerance and quality of life for patients and demonstrates good safety, highlighting its high clinical application value. As a new strategy for heart failure management, this combined treatment offers an important direction for optimizing treatment outcomes and has broad implications for promotion.

Disclosure

The authors report no conflicts of interest in this work.

References

1. Dick SA, Epelman S. Chronic heart failure and inflammation: what do we really know? Circ Res. 2016;119(1):159–176. doi:10.1161/CIRCRESAHA.116.308030

2. Rogers C, Bush N. Heart failure: pathophysiology, diagnosis, medical treatment guidelines, and nursing management. Nurs Clin North Am. 2015;50(4):787–799. doi:10.1016/j.cnur.2015.07.012

3. Sinnenberg L, Givertz MM. Acute heart failure. Trends Cardiovasc Med. 2020;30(2):104–112. doi:10.1016/j.tcm.2019.03.007

4. Hoffman TM. Chronic heart failure. Pediatr Crit Care Med. 2016;17(8 Suppl 1):S119–23. doi:10.1097/PCC.0000000000000755

5. Skrzypek A, Mostowik M, Szeliga M, et al. Chronic heart failure in the elderly: still a current medical problem. Folia Med Cracov. 2018;58(4):47–56.

6. Docherty KF, Vaduganathan M, Solomon SD, et al. Sacubitril/valsartan: neprilysin inhibition 5 years after PARADIGM-HF. JACC Heart Fail. 2020;8(10):800–810. doi:10.1016/j.jchf.2020.06.020

7. Huang E, Bernard ML, Elise Hiltbold A, et al. Sacubitril/valsartan: an antiarrhythmic drug? J Cardiovasc Electrophysiol. 2022;33(11):2375–2381. doi:10.1111/jce.15670

8. Sauer AJ, Cole R, Jensen BC, et al. Practical guidance on the use of sacubitril/valsartan for heart failure. Heart Fail Rev. 2019;24(2):167–176. doi:10.1007/s10741-018-9757-1

9. McDonagh TA, Metra M, Adamo M, et al. 2021 ESC guidelines for the diagnosis and treatment of acute and chronic heart failure: developed by the task force for the diagnosis and treatment of acute and chronic heart failure of the European society of cardiology (ESC) with the special contribution of the heart failure association (HFA) of the ESC. Rev Esp Cardiol. 2022;75(6):523. doi:10.1016/j.rec.2022.05.005

10. Horodinschi RN, Bratu OG, Dediu GN, et al. Heart failure and chronic obstructive pulmonary disease: a review. Acta Cardiol. 2020;75(2):97–104. doi:10.1080/00015385.2018.1559485

11. Amdani S, Conway J, George K, et al. Evaluation and management of chronic heart failure in children and adolescents with congenital heart disease: a scientific statement from the American heart association. Circulation. 2024;150(2):e33–e50. doi:10.1161/CIR.0000000000001245

12. Shrestha J, Done S. An overview of chronic heart failure. Nurs Stand. 2023;38(8):43–49. doi:10.7748/ns.2023.e12049

13. Donal E, L’Official G, Kosmala W. New guidelines for managing chronic heart failure patients and new needs in echocardiography. Int J Cardiol. 2022;353:71–72. doi:10.1016/j.ijcard.2022.01.035

14. Lye M. Chronic heart failure–mechanisms and management. Scott Med J. 1997;42(5):138–140. doi:10.1177/003693309704200507

15. Asano R, Mathai SC, Macdonald PS, et al. Oxygen use in chronic heart failure to relieve breathlessness: a systematic review. Heart Fail Rev. 2020;25(2):195–205. doi:10.1007/s10741-019-09814-0

16. Heyse A, Manhaeghe L, Mahieu E, et al. Sacubitril/valsartan in heart failure and end-stage renal insufficiency. ESC Heart Fail. 2019;6(6):1331–1333. doi:10.1002/ehf2.12544

17. Fonseca C, Brito D, Ferreira J, et al. Sacubitril/valsartan: a practical guide. Rev Port Cardiol. 2019;38(5):309–313. doi:10.1016/j.repc.2018.10.008

18. Telesca M, De Angelis A, Donniacuo M, et al. Effects of sacubitril-valsartan on aging-related cardiac dysfunction. Eur J Pharmacol. 2024;978:176794. doi:10.1016/j.ejphar.2024.176794

19. Dargad RR, Prajapati MR, Dargad RR, et al. Sacubitril/valsartan: a novel angiotensin receptor-neprilysin inhibitor. Indian Heart J. 2018;70(Suppl 1):S102–s110. doi:10.1016/j.ihj.2018.01.002

20. Singh JSS, Burrell LM, Cherif M, et al. Sacubitril/valsartan: beyond natriuretic peptides. Heart. 2017;103(20):1569–1577. doi:10.1136/heartjnl-2017-311295

21. Słonina G, Zemleduch T, Kimla P, et al. Is it time for the practical application of biomarkers in chronic heart failure management? Pol Merkur Lekarski. 2022;50(296):73–77.

22. Fiore G, Suppress P, Triggiani V, et al. Neuroimmune activation in chronic heart failure. Endocr Metab Immune Disord Drug Targets. 2013;13(1):68–75. doi:10.2174/1871530311313010009

23. Ahmad T, Fiuzat M, Felker GM, et al. Novel biomarkers in chronic heart failure. Nat Rev Cardiol. 2012;9(6):347–359. doi:10.1038/nrcardio.2012.37

24. Hale ZE, Prichett L, Jandu S, et al. Sacubitril-valsartan vs ACE/ARB in pediatric heart failure: a retrospective cohort study. J Heart Lung Transplant. 2024;43(5):826–831. doi:10.1016/j.healun.2024.01.012

25. Velicki L, Popovic D, Okwose NC, et al. Sacubitril/valsartan for the treatment of non-obstructive hypertrophic cardiomyopathy: an open label randomized controlled trial (SILICOFCM). Eur J Heart Fail. 2024;26(6):1361–1368. doi:10.1002/ejhf.3291

26. Yang X, Jin J, Cheng M, et al. The role of sacubitril/valsartan in abnormal renal function patients combined with heart failure: a meta-analysis and systematic analysis. Ren Fail. 2024;46(1):2349135. doi:10.1080/0886022X.2024.2349135

27. Myhre PL, Vaduganathan M, Claggett B, et al. B-type natriuretic peptide during treatment with sacubitril/valsartan: the PARADIGM-HF trial. J Am Coll Cardiol. 2019;73(11):1264–1272. doi:10.1016/j.jacc.2019.01.018

28. Berg DD, Samsky MD, Velazquez EJ, et al. Efficacy and safety of sacubitril/valsartan in high-risk patients in the PIONEER-HF Trial. Circ Heart Fail. 2021;14(2):e007034. doi:10.1161/CIRCHEARTFAILURE.120.007034

29. Matsumoto S, McMurray JJV, Nasu T, et al. Relevant adverse events and drug discontinuation of sacubitril/valsartan in a real-world Japanese cohort: REVIEW-HF registry. J Cardiol. 2024;84(2):133–140. doi:10.1016/j.jjcc.2023.11.005

30. Kang H, Zhang J, Zhang X, et al. Effects of sacubitril/valsartan in patients with heart failure and chronic kidney disease: a meta-analysis. Eur J Pharmacol. 2020;884:173444. doi:10.1016/j.ejphar.2020.173444

31. Fuzaylova I, Lam C, Talreja O, et al. Sacubitril/Valsartan (Entresto^®)-Induced Hyponatremia. J Pharm Pract. 2020;33(5):696–699. doi:10.1177/0897190019828915

Introduction

Pseudomonas aeruginosa is ubiquitous in nature and a well-known opportunistic pathogen in hospitalized immunocompromised patients. P. aeruginosa can infect a range of systems and tissues, causing various types of inflammation and infections such as bacteremia, septicemia, septic pyemia, pneumonia, bronchitis, diarrhea, keratitis, and skin and wound infections.^1,2 Multidrug-resistant P. aeruginosa poses a serious clinical threat, and the development of carbapenem resistance is expected to exacerbate this situation.³ Carbapenem-resistant P. aeruginosa (CRPA) has been categorized as a high-priority threat by the World Health Organization’s updated bacterial priority pathogen List (BPPL) 2024.⁴

Resistance to carbapenems in P. aeruginosa is generally due to a combination of mechanisms, including deficiency of the OprD porin, overexpression of efflux pumps, intrinsic chromosomally encoded AmpC β-lactamase, and production of carbapenemase.^2,5,6 The production, activity, transferability, and prevalence of metallo-β-lactamases (MBLs), particularly members of imipenem-type and Verona integron-encoded MBL families, are the most clinical significance.⁷ A P. aeruginosa strain carrying the MBL-coding gene bla_IMP-45, which was first reported in 2014, was isolated from a canine in Beijing.⁸ Recently, 23 bla_IMP-45-carrying conjugative IncP-2 megaplasmids, carried by Pseudomonas spp. from diverse genetic backgrounds, have been reported worldwide.^8–19 Among them, P. aeruginosa clone ST508 caused an outbreak of nosocomial infections at Hospital HS (Shanghai, China) from January to September 2015.¹²

Heteroresistance, which can generally be defined as the presence of subpopulations of cells within a culture of a single bacterial isolate with higher levels of antibiotic resistance, is a phenomenon that causes unreliability in antimicrobial susceptibility testing.²⁰ It is widely accepted that heterogeneous resistance arises from the unstable amplification of resistance genes, likely involving complex interactions between mobile genetic elements, including plasmids, resistance gene cassettes, transposons, and insertion sequences. Heteroresistance to carbapenem has been well-documented in current clinical isolates of Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii.^21–23 Few studies have investigated the mechanisms of carbapenem-heteroresistant P. aeruginosa, which have shown the involvement of reduced OprD porin expression, efflux pump system overexpression, and biofilm formation.²⁴ The mechanisms by which MBLs and MBL-harboring plasmids mediate carbapenem-resistant P. aeruginosa remain unclear.

This study pioneers the exploration of imipenem resistance mechanisms in P. aeruginosa mediated by MBLs-encoding genes and their plasmid-borne transmission pathways.Heterogeneous resistant strains carrying the bla_IMP-45 gene showed a 32-fold increase in the maximum permissible growth concentration of 256 compared to non-heterogeneous resistant strains not carrying bla_IMP-45. The maximum allowable growth concentration of 256 was 32-fold higher in the former than in the latter and 2-fold higher in the latter than in the heterogeneously resistant strain carrying bla_IMP-45. Plasmid sequencing analysis showed that the genetic environment of bla_IMP-45 was consistent, and there was no variation in the promoter region. Pre-exposure of the strains to a low concentration of imipenem for 24h caused an increase in the expression and copy number of bla_IMP-45 in the heterogeneous resistant strains HN232 and HN41, except that the increase was more pronounced in the former.

Material and Methods

Bacteria Strains and Identification

The following non-duplicate clinical P. aeruginosa isolates were obtained from sputum or wound secretion samples collected mainly from two different surgical wards at the People’s Hospital of Sanya City, Hainan Province, between 2013 and 2014: HN41, HN66, HN67, HN125, HN148, and HN232. Bacterial species were initially identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF; Bruker,Germany). The criterion for strain inclusion was (IMP) readings of 1–3 mg/L (described below).

Genome Sequencing, Resistance Genes, Species Confirmation and MLST

Genomic DNA was extracted using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) and sequenced on an advanced Illumina NovaSeq 6000 platform.A double-ended 150 bp (PE150) strategy was adopted. Average sequencing depth of andgt;100 × per sample, data volume of ≥6 GB, QC requirements: Q30 >85% (base-balanced library), and compliance with Illumina standard QRP39. Raw data was QC controlled by FastQC v0.12.1 and low-quality reads (Phred score andlt;20) and adapter sequences were filtered using Trimmomatic v0.39. The resistance gene profiles of the strains were analyzed using ResFinder (http://genepi.food.dtu.dk/resfinder) and multilocus sequence typing (MLST) was performed using the tool available at https://cge.food.dtu.dk/services/MLST/. Species identification was confirmed using SpeciesFinder at the Center for Genomic Epidemiology (http://genomicepidemiology.org/services/).

Antimicrobial Susceptibility Testing and Screening of HR Strains

Antimicrobial susceptibility testing was performed using the broth microdilution method with customized microtiter plates containing vacuum-dried antibiotics (BD,Phoenix NMIC-413, USA). The MIC values were interpreted according to the 2024 Clinical and Laboratory Standards Institute (CLSI) guidelines.²⁵ E. coli strain ATCC 25922 was used as a quality control. Initial screening of HR strains was conducted using E-test strips (BioMérieux, France). Suspicion of heterogeneous resistance was based on the presence of resistant clones inside the zone of inhibition or unclear boundaries.

Population Analysis Profile (PAP) Test

Bacterial suspensions containing 10⁻⁸–10⁻⁴ colony-forming units (CFU)/mL were prepared from a 0.5 McFarland standard (~ 0.1×10⁸ CFU/mL). A 100-μL aliquot of each bacterial suspension was spotted in triplicate onto fresh gradient-diluted Mueller-Hinton agar (MHA) plates containing IMP at the following concentrations: 0, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 mg/L. The plates were incubated at 37°C for 24h and the number of CFUs at each concentration was used to draw the PAP curves. The analysis was performed in triplicate with carbapenem-susceptible P. aeruginosa ATCC27853 as the control strain.²⁶

Measurement of HN232 Resistance Frequency Under IMP Pre-Exposure

To investigate the effect of different IMP pre-exposure concentrations on the frequency of resistant clones, five different pre-exposure concentrations of IMP (0.5, 1, 4, 32, and 128 mg/L) were added to bacterial suspensions of HN232 activated for growth in Mueller-Hinton broth (MHB) and incubated for 24h at 37°C. Pre-exposure to 0 mg/L IMP served as a negative control. A 100 μL aliquot of each bacterial suspension was spotted in triplicate onto fresh gradient-diluted Mueller-Hinton agar (MHA) plates containing IMP at the following concentrations: 0, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256, and 512 mg/L. The plates were incubated at 37°C for 24h and count the number of CFUs at each concentration (Figure 1). The analysis was performed in triplicate. The percentage of bacterial regrowth to IMP was calculated by dividing the number of CFUs on the IMP-containing MHA plates by the number of CFUs on the MHA plates without IMP.²⁷

Figure 1 Pre-exposed with IMP to measure HN232 resistance frequency.

Determination of the bla_IMP-45 Genetic Environment in Plasmids HN232 and HN41

The complete genomes of HN232 and HN41 were sequenced by combining the data obtained from a whole-genome shotgun strategy using the Ion Torrent Personal Genome Machine system (Life Technologies, USA) and Pacific Biosciences RSII DNA sequencing platform (PacBio, USA). The contigs were assembled using the Hierarchical Genome Assembly Process version 3.0. Sequence annotation, open reading frame prediction, and pseudogene identification of plasmids were performed using RAST 2.0 (https://rast.nmpdr.org/). Annotation of resistance genes, mobile elements, and other features was carried out using the following online databases: ResFinder (http://genepi.food.dtu.dk/resfinder), ISfinder (https://www-is.biotoul.fr/index.php), INTEGRALL (http://integrall.bio.ua.pt/?) and the Tn Number Registry (http://www.ucl.ac.uk/eastman/research/departments/microbial-diseases/tn). Gene organization diagrams were constructed using Inkscape 0.48.1 (http://inkscape.org).

qRT-PCR Determination of Gene Expression

To study the effect of local amplification of plasmid-derived bla_IMP-45 on the development of heterogeneous drug resistance, the IMP-HR strains HN232 and HN41 were treated with different pre-exposure concentrations of IMP. qRT-PCR was used to examine the expression of bla_IMP-45, replication initiation protein A (repA), and the chromosomal housekeeping gene ecfX. The mRNA levels of bla_IMP-45 and repA were expressed as fold-increases relative to that of ecfX, which served as the control.

For each strain, three independent cultures were exposed to a gradient of six IMP concentrations. After a 24h pre-exposure, the cultures were grown in MHB. The bacteria were collected and total RNA was extracted using an RNeasy R Mini Kit (Qiagen, Germany). The RNA sample concentrations were measured using a NanoDrop ND-1000 spectrophotometer. Reverse transcription of RNA was performed using the Prime Script RT Reagent Kit (Takara Bio, Japan) in accordance with the manufacturer’s instructions. The incubation conditions for cDNA synthesis were 37°C for 15min, 85°C for 5min, and 4°C for 5min. Primers for the amplification of bla_IMP-45, plasmid-derived repA and ecfX were designed using the online tool (https://sg.idtdna.com/. All real-time PCRs were performed using a Bioer Quant Gene 9600 device. Each qRT-PCR reaction contained 5 µL 2 × SYBR Green (TIANGEN, China), 0.5 µL forward and reverse primers, 1 µL cDNA, and 3 µL RNase-free ddH₂O (Takara Bio, Japan). The cycling parameters were as follows: 95°C for 20s, followed by 40 cycles of 95°C for 10s, 55°C for 10s, and 72°C for 20s. Relative gene expression was calculated using the 2^−ΔΔCt method. At least three biological and technical replicates were performed for each set of experiments. The primers used are listed in Table S in supplementary material.

ddPCR Determination of Plasmid Copy Number

To study the copy numbers of bla_IMP-45 and repA in HN232 and HN41, ddPCR assays comprising of three reactions were performed. The reaction system comprised 10 µL of 2×ddPCR Supermix for Probes (no dUTP) (Bio-Rad, USA), 4 µL of template DNA, 0.4 µL of forward and reverse primers, 0.4 µL of probe, and 4.8 µL of ddH₂O, in a final reaction volume of 20 µL. Microdroplets of oil-encapsulated bacteria were generated using an automated microdroplet generator (Bio-Rad). Thermal cycling conditions using a Bio-Rad C1000 Touch™ Thermal Cycler were 95°C for 30s; 40 cycles at 95°C for 30s and 60°C for 30s; 98°C for 10min; and 4°C for 5min. The reaction data were examined using a QX200™ Droplet Analyzer and Bio-Rad Quanta Soft software for Poisson distribution analysis and calculation of absolute copy numbers. At least three biological and technical replicates were performed for each set of experiments. The primers used are listed in Supplementary material in Table S.

Nucleotide Sequence Accession Numbers

The complete sequences of HN41 chromosome, pHN41-MDR, HN232 chromosome, and pHN232-MDR were submitted to GenBank under accession numbers CP173219, CP173220, CP151543, and CP151544, respectively.

Results

Genomic Backgrounds and Antimicrobial Susceptibility Phenotypes and Genotypes of the CRPA Strains

Among the six CRPA isolates, two belonged to the same sequence type ST (ST446), whereas the remaining four had unique STs, namely ST110, ST111, ST2631, and ST693.

All six strains were susceptible to aztreonam and colistin and resistant to cephalosporins, including ceftazidime, cefepime, and others including piperacillin/tazobactam, and meropenem. However, these strains showed varying degrees of sensitivity and resistance to IMP, fluoroquinolones (ciprofloxacin and levofloxacin) and amikacin. Regarding the response to IMP, HN66 and HN67 exhibited sensitivity, HN125 and HN232 showed intermediate susceptibility, and HN41 and HN148 exhibited resistance. All six strains showed MICs of 1–3 mg/L IMP on E-test strip readings (Table 1), with either fuzzy borders or visible colonies scattered within the inhibition zone (Supplementary material: Figure S), and were suspected to be IMP-heterogeneous.

Table 1 Characteristics of Six P. Aeruginosa Clinical Isolates Carrying blaIMP-45

Regarding the antibiotic resistance genotype, strains HN67 and HN125 were negative for bla_IMP-45 and positive for the presence of five resistance genes: aminoglycoside-3’-phosphotransferase gene aph (3’)-IIb, bla_{OXA-486/395/488/50/485}, bla_PAO-1, fosfomycin fosA, and catB7. These five genes were shared by all six isolates (excluding certain single-nucleotide polymorphisms within bla_{486/395/488/50}), suggesting that they were chromosomally located intrinsic resistance genes. Among the four bla_IMP-45-positive isolates, HN148 was found to carry a multidrug resistance operon and the following 14 resistance genes: rifampin gene arr-2; β-lactam genes bla_OXA-485, bla_PAO, bla_IMP-45 and bla_OXA-1; aminoglycoside genes aph (3’)-Ilb and aac (6’)-Ib3, chloramphenicol genes catB3 and catB7, fosfomycin gene (fosA), tetracycline gene cluster oprJ-tmexCD3, quinolone gene qnrVC1, trimethoprim gene dfrA22, and sulfonamide gene sul1. Compared with HN148, HN232 carries four additional resistance genes: aminoglycoside resistance gene armA, aph (3’)-Ia, macrolide-specific efflux pump gene msr(E), and macrolide phosphotransferase gene mph(E). Compared to HN232, HN66 and HN41 carried tet(C) and ciprofloxacin resistance protein-encoding crpP.

Screening and Confirmation of IMP-HR and IMP-NHR P. aeruginosa Strains

The heteroresistance non-inhibitory concentration (HNIC), heteroresistance inhibitory concentration (HIC), HIC/HNIC ratio, and frequency of resistant clones at the HNIC of IMP for the six isolates were determined using the PAP test (Table 2). Based on the classical definition of heteroresistance (presence of a subpopulation of resistant bacteria at ≥ 10⁻⁷ frequency that can grow in an antibiotic concentration ≥ 8-fold higher than the highest concentration affecting the growth of the main population),^28,29 strains HN66, HN148, and HN232 were classified as IMP-HR, HN41, HN67and HN125 as IMP-NHR (Figure 2).

Table 2 Validation of the Heteroresistant Status of P. Aeruginosa Strains Using the PAP Method

Figure 2 Population analysis profile (PAP) curves of six P. aeruginosa strains to IMP. Abbreviations: CFU, colony-forming units; HR, heterogeneous resistance; NHR, non-heterogeneous resistance.

Notably, in strains HN67 and HN125, which did not carry bla_IMP-45, the IMP HICs were 8 mg/L. In contrast, four of the bla_IMP-45-positive strains, HN41, HN148, HN66, and HN232, exhibited HICs that were significantly elevated by at least 32-fold (≤ 256 mg/L) compared to HN67 and HN125.

Impact of IMP Pre-Exposure on IMP-HR Strain HN232

As shown in Figure 3, at pre-exposure IMP concentrations of 1, 4, and 32 mg/L, the regrowth percentage and frequency of HN232 on plates containing a range of IMP concentrations showed an overall trend of initially increasing and then decreasing; however, they remained higher than those of the controls. At lower (0.5 mg/L) and higher (128 mg/L) IMP concentrations, the effect of pre-exposure on regrowth was not significant. The HICs of IMP for HN232 after pre-exposure to 0.5, 1, 4, 32, and 128 mg/L were 256, 256, 256, 128, and 128 mg/L, respectively, consistent with the HIC observed in the PAP experiment without pre-exposure. These results indicated that pre-exposure treatment did not affect HIC, but selectively increased the aggregation of resistant subpopulations at specific concentrations of IMP.

Figure 3 Impact of pre-exposure to IMP on the formation of IMP-resistant subpopulations. Schematic diagram illustrating the pre-exposure of HN232 to different doses of IMP-containing liquid medium for 24 h followed by regrowth on Mueller-Hinton agar plates containing a gradient of IMP concentrations (0–512 mg/L). The percentage of bacterial strains regrowing on IMP plates at different concentrations. Experiments were independently repeated three times. Error bars represent the standard error of the mean (n = 3).

At pre-exposure to 1 mg/L IMP, the frequencies of HN232 bacteria on regrowth plates (0.5, 1, 2, 4 and 8 mg/L IMP) were 7.3×10⁻⁶, 1.03×10⁻⁷, 9.2×10⁻⁶, 8.6×10⁻⁶, and 7.5×10⁻⁶, respectively. The percentage of bacteria regrown on these plates was > 1. At pre-exposure to 4 mg/L IMP, the percentages of regrowth bacteria on plates containing 0.5, 1, 2 and 4 mg/L IMP were also > 1, with frequencies of regrowth bacteria of 7.19×10⁻⁶, 7.79×10⁻⁶, 7.42×10⁻⁶, and 7.24×10⁻⁶, respectively. However, this effect was not as evident at a pre-exposure IMP concentration of 1 mg/L, indicating that exposure to low IMP concentrations (1 and 4 mg/L) was sufficient to generate an IMP-resistant subpopulation that grew on plates containing ≥ 4 mg/L IMP (intermediate resistance) within a relatively short period. At pre-exposure to 32 mg/L IMP, the percentages of HN232 bacteria on regrowth plates at 0.5 and 1 mg/L IMP were also > 1, with frequencies of 7.1×10⁻⁶ and 7.35×10⁻⁶, respectively. In contrast, at 128 mg/L pre-exposure, the percentage of regrowth bacteria across all plates was < 1, suggesting that high concentrations of IMP may exert bactericidal effects and cause toxicity, thereby inhibiting bacterial growth.

Genetic Environment and bla_IMP-45 Promoter in HN232 and HN41

Genomic sequencing and assembly revealed the presence of 433,528-kb and 449,083-kb closed circular DNAs carrying bla_IMP-45 in HN232 and HN41 strains, which were hereafter designated as pHN232-MDR and pHN41-MDR, respectively. Plasmid pHN232-MDR encompassed 96% of the pHN41-MDR sequence and shared 100% nucleotide identity in the overlapping region. Both plasmids possessed the core characteristics of IncP-2 group megaplasmids (> 300 kb in size), including genes essential for replication (repA2, 1188 bp) and partition (parB2-parAB), and gene clusters coding for conjugation (tra), pilus assembly (pil), chemotaxis (che), and tellurium resistance (ter) functions in the plasmid backbone (data not shown).

The genetic environment adjacent to bla_IMP-45 in both HN232 and HN41 was found to be associated with Tn1403-based Tn6485-like transposons (Figure 4). Tn1403 is a Tn3-family transposon that was initially identified in a clinical P. aeruginosa isolate from the USA in the 1970s.³⁰ It has a core backbone composed of inverted repeat left (IRL), tnpA (transposase), tnpR (resolvase), res (resolution site), sup (sulfate permease), uspA (universal stress protein), dksA (RNA polymerase-binding transcription factor), yjiK (hypothetical protein), and inverted repeat right (IRR), with insertion sequence elements In28 and Tn5393c inserted into res and dksA, respectively. Tn1403 and its close derivatives Tn6060, Tn6061, Tn6217, Tn6249, and Tn6286,^19,31–33 and the recently reported Tn6485a-h in Pseudomonas, often contain insertions of different foreign entities (eg, integrons and other transposons),^9,12,15,16 serving as important vehicles for the transmission of resistance genes.

Figure 4 Genetic environment of blaIMP-45 in pHN232-MDR and pHN41-MDR, and comparison with Tn1403. Genes are denoted by arrows. Colors of genes, mobile elements and other features indicate their functional classification. Shading denotes regions of homology (> 95% nucleotide identity). Numbers in brackets indicate the nucleotide positions within the corresponding plasmids. Pchybrid 1 (PcH1) and P2 promotors within In786.

Tn6485-like transposon Tn7445, found in both pHN232-MDR and pHN41-MDR, was 32,724 bp in length, with 38-bp IRLs and IRRs identical to those of Tn1403 and bordered by the same 5-bp TCTCA direct repeat, that is, target site duplications that indicate acquisition of Tn7445 by transposition. Compared to Tn1403, Tn7445 undergoes several biological events.

1. In28 was replaced with In786, which had a gene cassette array of aacA4cr (aminoglycoside and quinolone resistance)-bla_IMP-45–gcu35 (unknown function)-bla_OXA-1 (β-lactamase resistance).
2. Evolution of In786 into a complex class 1 integron via acquisition of the Tn1548-associated region extending from the ISCR1 element to IS26, which harbors armA and the macrolide resistance operon msr(E)–mph(E).
3. Accumulation of two entities, namely the IS26-aphA1-IS26 aminoglycoside resistance unit and Tn6309, which carries tetA(C) downstream of the Tn1548-associated region. Furthermore, these three entities overlapped at one of the terminal IS26 elements, strongly suggesting that they were acquired via IS26-mediated homologous recombination, rather than transposition.
4. Loss of most of the Tn1403 backbone (Δres-sup-uspA-dksA-yjiK) was replaced by five cryptic genes.

Relative Expression Levels and Copy Numbers of the bla_IMP-45 and repA Genes in HN232 and HN41

In the IMP-HR strain HN232, following pre-exposure to 1, 2, 8, 32, 128, and 512 mg/L IMP for 24h, the expression levels of repA relative to those of ecfX (1.36-, 1.23-, 1.17-, 0.96-, 0.73-, and 0.38-fold, respectively) suggested that plasmid replication was slightly induced at concentrations ≤ 8 mg/L, but was inhibited at concentrations ≥ 32 mg/L (Figure 5A). The expression levels of bla_IMP-45 relative to those of ecfX in the same range of IMP concentrations (20.00, 3.95, 1.59, 1.31, 1.03, and 0.53-fold, respectively) indicated that bla_IMP-45 expression might be induced by pre-exposure to ≤ 8 mg/L IMP, especially at 1 and 2 mg/L.

Figure 5 Quantitative real-time PCR and droplet digital PCR analyses of blaIMP-45 in strains HN41 and HN232. (A and B) Expression of blaIMP-45 and repA relative to ecfX in IMP-HR strain HN232 and IMP-NHR strain HN41 exposed to various concentrations of IMP. (C) Copy numbers of blaIMP-45 and repA in HN232 and HN41 exposed to various concentrations of IMP. Experiments were independently repeated three times. Error bars represent the standard error of the mean (n = 3).

In IMP-NHR strain HN41, following exposure to 1, 2, 8, 32, 128, and 512 mg/L IMP, the expression levels of repA relative to those of ecfX (0.77-, 0.48-, 0.23-, 0.16-, 0.08-, and 0.02-fold) suggested that plasmid replication was inhibited at all concentrations, especially at ≥ 8 mg/L (Figure 5B). The expression levels of bla_IMP-45 relative to those of ecfX at the same range of IMP concentrations (0.95-, 0.6-, 0.2-, 0.1-, 0.07-, and 0.02-fold, respectively) suggested that pre-exposure to 1 and 2 mg/L slightly increased the expression of bla_IMP-45, all IMP concentrations inhibited the replication of the plasmid itself and the expression of bla_IMP-45, especially at concentrations ≥ 8 mg/L.

The absolute copy numbers of bla_IMP-45 and repA in HN232 and HN41 under pre-exposure IMP concentrations of 1 mg/L, 128 mg/L, and 512 mg/L were determined using ddPCR (Figure 5C). While the ratio of bla_IMP-45 to repA varied between 2.8–3.8, the greatest difference in absolute copy numbers was observed at 1 mg/mL IMP in HN232, in which the absolute copy number of bla_IMP-45 exceeded that of repA by > 3,000 copies, providing strong evidence for the independent replication of bla_IMP-45 resulting from unstable gene amplification. In HN41, the bla_IMP-45 copy number was also considerably higher than that of repA, but was not induced to the same extent as in HN232.

Discussion

The presence of carbapenemases often leads to an MDR phenotype in P. aeruginosa, with frequent reports of strains harboring the MBL gene bla_IMP-45 in recent years.^{8,11,12,16,34,35} The STs of P. aeruginosa isolates include ST235, ST274, ST277, ST308, ST357, ST369, ST389, ST508, ST708, ST1420, and ST3014,^8,12,15,16 among these, ST508 and ST463 are high-risk clones involved in nosocomial infection outbreaks. To the best of our knowledge, ST693 and ST2631 identified in this study are novel bla_IMP-45-carrying STs in P. aeruginosa. Phenotypically, the MIC values of meropenem for the six P. aeruginosa strains in this study were all > 8 mg/L (Table 1), which is above the resistance breakpoint recommended by CLSI 2024.²⁵ CRPA isolates are generally defined by resistance to ≥ one carbapenems with antipseudomonal activity (doripenem, IMP, or meropenem);³⁶ therefore, all six of our strains were classified as CRPA. However, the four bla_IMP-45-carrying strains showed different MIC values (≤ 0.25 to > 8 mg/L) for IMP (Table 1). This may be attributable to the different pharmacokinetic traits of IMP and meropenem; the anti-bactericidal activity and killing speed of IMP against P. aeruginosa are significantly greater than those of meropenem.³⁷ Three of the bla_IMP-45-carrying isolates (HN66, HN148, and HN232) were confirmed to be IMP-HR; however, whether these strains are also HR to meropenem requires further investigation.In one study, rates of CRPA heteroresistance to meropenem and IMP have been reported 72.5% and 54.3%, respectively, indicating variability.³⁸

The results of PAP testing and IMP pre-exposure experiments revealed that the HIC of bla_IMP-45-carrying IMP-HR isolates could be increased 32-fold (up to 256 mg/L) relative to that of bla_IMP-45-negative IMP-HR isolates. Additionally, pre-exposure of the strains to low concentrations of IMP (1 and 4 mg/L) led to an increased frequency of regrowth on plates containing IMP concentrations associated with intermediate susceptibility and resistance. Both factors could contribute to the difficulty in completely eradicating IMP-HR strains during clinical treatment, thereby increasing the likelihood of infections evolving into refractory infections and treatment failure.

Examination of the genetic environment of bla_IMP-45 in the CRPA isolates revealed that this gene is located in the second gene cassette of In786 within the Tn1403-derived transposon, which resides in the Inc-P2 megaplasmid. The expression of bla_IMP-45 was governed by the same promoter in both IMP-HR HN232 and IMP-NHR HN41, excluding the possibility that the observed variation in gene expression levels was due to genetic and environmental differences. In contrast to the high genetic diversity of the genetic background of host strains, the genetic environment of bla_IMP-45 is relatively monotonous. Except for two cases of bla_IMP-45 found on the chromosomes of P. aeruginosa strains PA59 and M140A,³⁹ all the fully sequenced bla_IMP-45-carrying plasmids belonged to the IncP-2 group. Prior to 2013, the bla_IMP-45-encoding plasmid pOZ176 (500 kb) was the only fully sequenced IncP-2 plasmid with bla_IMP-45 residing in the second gene cassette located within In244 (aacA4’-bla_IMP-9–aacA4’).⁸ Subsequently, in 2017 and 2018, two IncP-2 plasmids, pSY153-MDR¹⁸ and pBM413,¹⁶ were reported in Pseudomonas putida and P. aeruginosa, respectively. Both were shown to carry bla_IMP-45 (the G214S variant of bla_IMP-9) in In786. To date, at least 23 bla_IMP-45 plasmids have been reported,^8–19 and phylogenetic analysis has revealed that most plasmids carrying bla_IMP-45 cluster into a distinct sublineage that also contains the full pOZ176 sequence, facilitating the dissemination of bla_IMP-45 among genetically diverse P. aeruginosa strains.Despite all carrying the identical bla_IMP-45 gene, the isolates displayed ≥ 32-fold variation in imipenem MICs (each exceeding the clinical resistance breakpoint).This divergence, in the absence of any promoter or cassette polymorphism, indicates that the variable resistance levels are not encoded by the carbapenemase itself but are driven by the following mechanisms:inducible local amplification of the gene, differential plasmid replication control, and additional non-specific resistance traits such as efflux-pump overexpression.

In-depth mechanistic experiments indicated that plasmid replication in the IMP-NHR strain HN41 was suppressed at all IMP concentrations compared with chromosomal replication. In contrast, in the IMP-HR strain HN232, plasmid replication was inhibited at high concentrations of IMP but was roughly equivalent to chromosomal replication at concentrations between 1 and 32 mg/L. A common feature observed in both strains was the increased expression of bla_IMP-45 at IMP concentrations of 1 and 2 mg/L, with the fold increase being more pronounced in the HR strain. Absolute copy number quantification of bla_IMP-45 and repA using ddPCR further revealed that low concentrations of IMP induced an increase in bla_IMP-45 copy number in both HR and NHR isolates, albeit to a greater extent in the IMP-HR strain HN232. These findings support the hypothesis that bla_IMP-45 can undergo localized, unstable gene amplification independent of plasmid replication, leading to increased gene copy number and expression, which may play a major role in heteroresistance. Additionally, these findings suggest that plasmid replication and localized amplification of bla_IMP-45 are regulated by complex factors in both the strains. In comparison, plasmid replication and copy number may be more tightly controlled in IMP-NHR strain HN41.

Mobile genetic elements, such as IS26 and transposons, have been reported to mediate the unstable amplification of adjacent antibiotic resistance genes. Wei et al reported that after pre-exposure to sublethal doses of meropenem or tobramycin, IS26 mediated rapid and stable amplification of bla_KPC-2, leading to carbapenem resistance in clinical E. coli strains.²⁷ In A. baumannii, amplification of five resistance genes within the aadB region of plasmids has been reported to cause heteroresistance to tobramycin.¹³ Similarly, in Salmonella enterica serovar Typhimurium, gene amplification at the chromosomal pmrD locus is associated with heteroresistance to colistin.¹² Whether the unstable downstream amplification of bla_IMP-45 observed in our study was driven by IS26 or a Tn1403-like transposon remains to be confirmed.

While this study provides compelling evidence for the role of the bla_IMP-45-bearing plasmid and its inducible amplification in driving IMP heteroresistance in P. aeruginosa, it is important to acknowledge a potential limitation regarding the temporal scope of the isolates investigated. The clinical strains analyzed in this work were collected between 2013 and 2014. Antimicrobial resistance profiles and the prevalence of specific resistance mechanisms, including carbapenemases like IMP variants, are known to evolve dynamically over time in response to selective antibiotic pressure and infection control practices. Consequently, the specific resistance landscape and the relative abundance of bla_IMP-45 or similar determinants observed in this cohort may not fully represent the current clinical epidemiology of P. aeruginosa carbapenem resistance or the predominant mechanisms underpinning heteroresistance in contemporary settings. Future studies incorporating more recent clinical isolates would be valuable to confirm the ongoing relevance of the plasmid-mediated amplification mechanism described here and to assess potential shifts in the genetic drivers of IMP heteroresistance.

Conclusion

Carbapenem antibiotics remain the first-line treatment for P. aeruginosa infection. IncP-2 conjugative megaplasmids still serve as predominant vehicles for the dissemination of bla_IMP-45 among P. aeruginosa isolates. Our results indicate that, in IMP-HR bla_IMP-45-carrying P. aeruginosa, short-term (24 h) exposure to IMP concentrations as low as 1 mg/L can induce unstable localized gene amplification of plasmid-derived bla_IMP-45 in the frequency of occurrence of resistant sub populations. These findings emphasize the importance of rational antibiotic use in clinical treatment.

Ethics Statement

Human specimens were acquired with the patient’s consent. The use of human specimens and all related experimental protocols was reviewed and approved by the Ethics Committee of the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, in accordance with the medical research regulations of the Ministry of Health of China.The strain was routinely preserved in our laboratory repository, no patient interventions or additional sample collections were conducted. This study adhered to the guidelines outlined in the Declaration of Helsinki.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (grant number: L2124006).

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.

References

1. Si T, Wang A, Yan H. et al. Progress in the Study of Natural Antimicrobial Active Substances in Pseudomonas aeruginosa. Molecules. 2024;29(18):4400. doi:10.3390/molecules29184400

2. Alimoghadam S, Eslami A, Alimoghadam R, et al. The frequency of AmpC overproduction, OprD downregulation and OprM efflux pump expression in Pseudomonas aeruginosa: a comprehensive meta-analysis. J Global Antimicrob Resist. 2024;39:159–169. doi:10.1016/j.jgar.2024.08.014

3. Maiga A, Ampomah-Wireko M, Li H, et al. Multidrug-resistant bacteria quorum-sensing inhibitors: a particular focus on Pseudomonasaeruginosa. Eur J Med Chem. 2024;281:117008. doi:10.1016/j.ejmech.2024.117008

4. Jesudason T. WHO publishes updated list of bacterial priority pathogens. Lancet Microbe. 2024;5(9):100940. doi:10.1016/j.lanmic.2024.07.003

5. Wu W, Huang J, Xu Z. Antibiotic influx and efflux in Pseudomonas aeruginosa: regulation and therapeutic implications. Microb Biotechnol. 2024;17(5). doi:10.1111/1751-7915.14487

6. Ferous S, Anastassopoulou C, Pitiriga V, Vrioni G, Tsakris A. Antimicrobial and Diagnostic Stewardship of the Novel β-Lactam/β-Lactamase Inhibitors for Infections Due to Carbapenem-Resistant Enterobacterales Species and Pseudomonas aeruginosa. Antibiotics. 2024;13(3):285. doi:10.3390/antibiotics13030285

7. Tenover FC, Nicolau DP, Gill CM. Carbapenemase-producing Pseudomonas aeruginosa–an emerging challenge. Emerging Microbes Infect. 2022;11(1):811–814. doi:10.1080/22221751.2022.2048972

8. Wang Y, Wang X, Schwarz S, et al. IMP-45-producing multidrug-resistant Pseudomonas aeruginosa of canine origin. J Antimicrob Chemother. 2014;69(9):2579–2581. doi:10.1093/jac/dku133

9. Fang Y, Wang N, Wu Z, et al. An XDR Pseudomonas aeruginosa ST463 Strain with an IncP-2 Plasmid Containing a Novel Transposon Tn6485f Encoding bla(IMP-45) and bla(AFM-1) and a Second Plasmid with Two Copies of bla(KPC-2). Microbiol Spectrum. 2023;11(1):e0446222. doi:10.1128/spectrum.04462-22

10. Dong N, Liu C, Hu Y, et al. Emergence of an Extensive Drug Resistant Pseudomonas aeruginosa Strain of Chicken Origin Carrying bla(IMP-45), tet(X6), and tmexCD3-toprJ3 on an Inc(pRBL16) Plasmid. Microbiol Spectrum. 2022;10(6):e0228322. doi:10.1128/spectrum.02283-22

11. Wang N, Zheng X, Leptihn S, et al. Characteristics and phylogenetic distribution of megaplasmids and prediction of a putative chromid in Pseudomonas aeruginosa. Comput Struct Biotechnol J. 2024;23:1418–1428. doi:10.1016/j.csbj.2024.04.002

12. Zhang X, Wang L, Li D, et al. An IncP-2 plasmid sublineage associated with dissemination of blaIMP-45 among carbapenem-resistant Pseudomonas aeruginosa. Emerging Microbes Infect. 2021;10(1):442–449. doi:10.1080/22221751.2021.1894903

13. Urbanowicz P, Bitar I, Izdebski R, et al. Epidemic Territorial Spread of IncP-2-Type VIM-2 Carbapenemase-Encoding Megaplasmids in Nosocomial Pseudomonas aeruginosa Populations. Antimicrob Agents Chemother. 2021;65(4). doi:10.1128/AAC.02122-20.

14. Zhou D, Song Y, Dai E, et al. Plasmids of novel incompatibility group IncpRBL16 from Pseudomonas species. J Antimicrob Chemother. 2020;2020:1.

15. Zhang Y, Meng L, Guan C, Zhou Y, Peng J, Liang H. Genomic characterisation of clinical Pseudomonas aeruginosa isolate PAG5 with a multidrug-resistant megaplasmid from China. J Global Antimicrob Resist. 2020;21:130–131. doi:10.1016/j.jgar.2020.03.012

16. Liu J, Yang L, Chen D, et al. Complete sequence of pBM413, a novel multidrug resistance megaplasmid carrying qnrVC6 and bla IMP-45 from pseudomonas aeruginosa. Int J Antimicrob Agents. 2018;51(1):145–150. doi:10.1016/j.ijantimicag.2017.09.008

17. Zhou L, Yang C, Zhang X, et al. Characterization of a novel Tn6485h transposon carrying both blaIMP-45 and blaAFM-1 integrated into the IncP-2 plasmid in a carbapenem-resistant Pseudomonas aeruginosa. J Global Antimicrob Resist. 2023;35:307–313. doi:10.1016/j.jgar.2023.10.010

18. Yuan M, Chen H, Zhu X, et al. pSY153-MDR, a p12969-DIM-related mega plasmid carrying bla(IMP-45) and armA, from clinical Pseudomonas putida. Oncotarget. 2017;8(40):68439–68447. doi:10.18632/oncotarget.19496

19. Xiong J, Alexander DC, Ma JH, et al. Complete Sequence of pOZ176, a 500-Kilobase IncP-2 Plasmid Encoding IMP-9-Mediated Carbapenem Resistance, from Outbreak Isolate Pseudomonas aeruginosa 96. Antimicrob Agents Chemother. 2013;57(8):3775–3782. doi:10.1128/AAC.00423-13

20. Martínez-López N, Vilas C, García MR. A birth–death model to understand bacterial antimicrobial heteroresistance from time-kill curves. Math Biosci. 2024;376:1.

21. Shields RK, Dorazio AJ, Tiseo G, et al. Frequency of cefiderocol heteroresistance among patients treated with cefiderocol for carbapenem-resistant Acinetobacter baumannii infections. JAC-Antimicrob Resist. 2024;6(5). doi:10.1093/jacamr/dlae146.

22. Graffice E, Moates DB, Leal SM, Amerson-Brown M, Calix JJ. Epidemiological, Phylogenetic, and Resistance Heterogeneity Among Acinetobacter baumannii in a Large U.S. Deep South Healthcare system. Open Forum Infect Diseases. 2024;11(9). doi:10.1093/ofid/ofae458

23. Lin CK, Page A, Lohsen S, et al. Rates of resistance and heteroresistance to newer β-lactam/β-lactamase inhibitors for carbapenem-resistant Enterobacterales. JAC-Antimicrob Resist. 2024;6(2). doi:10.1093/jacamr/dlae048.

24. Xie X, Liu Z, Huang J, et al. Molecular epidemiology and carbapenem resistance mechanisms of Pseudomonas aeruginosa isolated from a hospital in Fujian, China. Front Microbiol. 2024;15. doi:10.3389/fmicb.2024.1431154

25. CLSIM100ED34-2024-Performance Standards for Antimicrobial Susceptibility Testing.pdf.

26. Li W-R, Zhang Z-Q, Liao K, et al. Pseudomonas aeruginosa heteroresistance to levofloxacin caused by upregulated expression of essential genes for DNA replication and repair. Front Microbiol. 2022;13. doi:10.3389/fmicb.2022.1105921

27. Wei DW, Wong NK, Song Y, et al. IS26 Veers Genomic Plasticity and Genetic Rearrangement toward Carbapenem Hyperresistance under Sublethal Antibiotics. mBio. 2021;13(1):e0334021. doi:10.1128/mbio.03340-21

28. El-Halfawy OM, Valvano MA. Antimicrobial Heteroresistance: an Emerging Field in Need of Clarity. Clin Microbiol Rev. 2015;28(1):191–207. doi:10.1128/CMR.00058-14

29. Nicoloff H, Hjort K, Andersson DI, Wang H. Three concurrent mechanisms generate gene copy number variation and transient antibiotic heteroresistance. Nat Commun. 2024;15(1). doi:10.1038/s41467-024-48233-0

30. Vézina G, Levesque RC. Molecular characterization of the class II multiresistance transposable element Tn1403 from Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1991;35(2):313–321. doi:10.1128/AAC.35.2.313

31. Chowdhury PR, Merlino J, Labbate M, Cheong EYL, Gottlieb T, Stokes HW. Tn 6060, a Transposon from a Genomic Island in a Pseudomonas aeruginosa Clinical Isolate That Includes Two Class 1 Integrons. Antimicrob Agents Chemother. 2009;53(12):5294–5296. doi:10.1128/AAC.00687-09

32. Di Pilato V, Pollini S, Rossolini GM. Tn6249, a New Tn 6162 Transposon Derivative Carrying a Double-Integron Platform and Involved with Acquisition of the bla VIM-1 Metallo-β-Lactamase Gene in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2015;59(3):1583–1587. doi:10.1128/AAC.04047-14

33. Coyne S, Courvalin P, Galimand M. Acquisition of multidrug resistance transposon Tn6061 and IS6100-mediated large chromosomal inversions in Pseudomonas aeruginosa clinical isolates. Microbiology. 2010;156(5):1448–1458. doi:10.1099/mic.0.033639-0

34. Kang Y, Xie L, Yang J, Cui J. Optimal treatment of ceftazidime-avibactam and aztreonam-avibactam against bloodstream infections or lower respiratory tract infections caused by extensively drug-resistant or pan drug-resistant (XDR/PDR) Pseudomonas aeruginosa. Front Cell Infect Microbiol. 2023;13. doi:10.3389/fcimb.2023.1023948

35. Lin H, Feng C, Zhu T, et al. Molecular Mechanism of the β-Lactamase Mediated β-Lactam Antibiotic Resistance of Pseudomonas aeruginosa Isolated From a Chinese Teaching Hospital. Front Microbiol. 2022;13:1.

36. Walters MS, Grass JE, Bulens SN, et al. Carbapenem-Resistant Pseudomonas aeruginosa at US Emerging Infections Program Sites, 2015. Emerging Infectious Diseases. 2019;25(7):1281–1288. doi:10.3201/eid2507.181200

37. Dreetz M, Hamacher J, Eller J, et al. Serum bactericidal activities and comparative pharmacokinetics of meropenem and imipenem-cilastatin. Antimicrob Agents Chemother. 1996;40(1):105–109. doi:10.1128/AAC.40.1.105

38. He J, Jia X, Yang S, et al. Heteroresistance to carbapenems in invasive Pseudomonas aeruginosa infections. Int J Antimicrob Agents. 2018;51(3):413–421. doi:10.1016/j.ijantimicag.2017.10.014

39. Ma W, Guo J, Deng C, et al. Characterization of the Chromosomally Located Metallo-β-Lactamase Genes bla(IMP-45) and bla(VIM-2) in a Carbapenem-Resistant Pseudomonas aeruginosa Clinical Isolate. Microbial Drug Resistance. 2024;30(10):422–431. doi:10.1089/mdr.2024.0059

Online retailer The Very Group has been taken over by US private equity firm Carlyle, bringing an end to more than 20 years under the ownership of the Barclay family.

The Littlewoods and Very owner confirmed that Washington-based Carlyle – a major lender to the firm – had become its controlling shareholder, with fellow lender Abu Dhabi-based investment fund, International Media Investments (IMI), continuing as a “key stakeholder”.

It ends a lengthy period under the ownership of the Barclay family, which has been forced to give up control of a raft of businesses in recent years, including The Daily Telegraph newspaper, The Ritz hotel in London and delivery company Yodel.

Carlyle and IMI first became lenders to The Very Group in 2021.

Robbie Feather, group chief executive of The Very Group, said: “This marks an important milestone for The Very Group as we move into an exciting new phase of growth.

“We are delighted to continue to partner with Carlyle and IMI.

“Their continued backing provides us with a stronger foundation to execute on our strategy, increase investment in technology and the customer experience and to build on the momentum across the business.”

The Barclay family, headed by identical twins Sir Frederick and the late Sir David, had bought the then Littlewoods catalogue business in 2002, before merging the business with Shop Direct in 2004, creating what in later years was rebranded as The Very Group.

Sir David died aged 86 in January 2021.

The brothers were among the UK’s most high-profile businessmen, whose assets made them billionaires at one stage and the family one of the richest in Britain.

But the family’s fortunes have apparently worsened in recent years and they lost control of the Telegraph newspapers after the family struggled to repay hefty loans.

Despite the change in control, Very Group – which sells electrical products, homeware and clothing – has delivered rising profits and just last month reported a 16% rise in underlying earnings to £307 million for the year to June 28 as sales reached £2.1 billion.

Mr Feather paid tribute to the Barclay family, recognising “their stewardship and contribution to the company over the past two decades”.

The company said: “Over this period and thanks to their leadership, The Very Group has grown significantly – evolving from a traditional catalogue business into one of the UK’s largest and most dynamic online retailers.

“The Barclay family pass on a business that is performing very strongly.”

Former chancellor Nadhim Zahawi is chairman of Very Group, having been appointed to the role in May last year.

LONDON, Nov. 10, 2025 /PRNewswire/ — Newmark Group, Inc. (Nasdaq: NMRK) (“Newmark” or “the Company”), a leading commercial real estate advisor and service provider to large institutional investors, global corporations, and other owners and occupiers, is pleased to announce the appointment of Andrew Wheldon and Matthew Bailey to its European Finance team, a move that underscores the Company’s commitment to expanding its world-class capital markets advisory in Europe. Wheldon, one of the UK’s leading real estate debt advisors, and Bailey, a premier structured finance investment banker, bring a combined 50-plus years of experience guiding complex transactions for top institutions. Their expertise further elevates Newmark’s global Debt, Equity and Structured Finance business across asset classes such as Residential, Offices, Industrial, Hospitality, Data Centres, Energy and powered land.

Michael Lehrman, President of UK and Europe, commented:
“Newmark’s continued investment in debt and structured finance is a cornerstone of our global capital markets strategy. Expanding this capability in Europe enables us to unlock cross-border capital flows, create deeper connectivity for our clients and deliver advisory solutions that match the sophistication of global capital markets today.”

Newmark has invested in expanding its Capital Markets capabilities worldwide. Wheldon and Bailey, who join as Managing Directors, follow the recent appointments of Andrew Allen, Hoong Wey Woon, Max Hagelstein and Phil Creed and data centre finance specialists Oliver Weston and Hamish Smith, among others, mark a significant expansion of Newmark’s Debt & Structured Finance offerings in Europe, led by Featherstone and Charlie Foster¹. In the U.S., Newmark has grown its debt origination market share by a multiple of six over the past decade².

Matthew Featherstone, Head of Debt & Structured Finance, UK & Europe, said:
“Andrew and Matthew’s specialist expertise in the living sector and structured finance significantly enhances our ability to advise on complex transactions and deliver innovative financing solutions for clients. Their experience strengthens our market position and deepens the bench of senior talent driving our growth across Europe.”

Andrew Wheldon joins Newmark as one of the UK’s foremost living sector debt advisors. With more than two decades of experience spanning capital advisory, real estate banking, development and asset management, he has played a pivotal role in shaping the UK living market. Andrew previously held senior roles at RBS, Lloyds Banking Group and CBRE Capital Advisors, where he led residential debt advisory, and later founded the UK arm of a pan-European capital advisory firm.

Matthew Bailey brings a distinguished track record in structured finance, specializing in commercial mortgage-backed securities, non-performing loans, restructurings and debt capital markets. Bailey has led the origination, structuring and execution of major structured real estate bond transactions across Europe.  He previously held senior roles at UBS, Commerzbank, HSBC Investment Bank and SitusAMC, Matthew graduated with an Applauded First from Oxford University where he received a BA in German and Philosophy and a Master’s in Philosophy.

About Newmark
Newmark Group, Inc. (Nasdaq: NMRK), together with its subsidiaries (“Newmark”), is a world leader in commercial real estate, seamlessly powering every phase of the property life cycle. Newmark’s comprehensive suite of services and products is uniquely tailored to each client, from owners to occupiers, investors to founders, and startups to blue-chip companies. Combining the platform’s global reach with market intelligence in both established and emerging property markets, Newmark provides superior service to clients across the industry spectrum. For the twelve months ended September 30, 2025, Newmark generated revenues of over $3.1 billion. As of September 30, 2025, Newmark and its business partners together operated from approximately 170 offices with over 8,500 professionals across four continents. To learn more, visit nmrk.com or follow @newmark.

Discussion of Forward-Looking Statements about Newmark
Statements in this document regarding Newmark that are not historical facts are “forward-looking statements” that involve risks and uncertainties, which could cause actual results to differ from those contained in the forward-looking statements. These include statements about the Company’s business, results, financial position, liquidity, and outlook, which may constitute forward-looking statements and are subject to the risk that the actual impact may differ, possibly materially, from what is currently expected. Except as required by law, Newmark undertakes no obligation to update any forward-looking statements. For a discussion of additional risks and uncertainties, which could cause actual results to differ from those contained in the forward-looking statements, see Newmark’s Securities and Exchange Commission filings, including, but not limited to, the risk factors and Special Note on Forward-Looking Information set forth in these filings and any updates to such risk factors and Special Note on Forward-Looking Information contained in subsequent reports on Form 10-K, Form 10-Q or Form 8-K.

Photo – https://mma.prnewswire.com/media/2817381/Matthew_Bailey_Andrew_Wheldon.jpg 
Logo – https://mma.prnewswire.com/media/1057994/Newmark_Group_Logo_v1.jpg

Terms

While we only use edited and approved content for Azthena
answers, it may on occasions provide incorrect responses.
Please confirm any data provided with the related suppliers or
authors. We do not provide medical advice, if you search for
medical information you must always consult a medical
professional before acting on any information provided.

Your questions, but not your email details will be shared with
OpenAI and retained for 30 days in accordance with their
privacy principles.

Please do not ask questions that use sensitive or confidential
information.

Read the full Terms & Conditions.

Terrorism takes its toll on Mozambique’s gas revenue

TotalEnergies’ return to Cabo Delgado will cost an extra US$4.5 billion – reflecting the price of terrorism and poor resource governance.

The decision by TotalEnergies to lift the force majeure on its Mozambique liquified natural gas (LNG) project marks the long-awaited restart of gas operations in Cabo Delgado. But the project’s four-and-a-half-year suspension due to the ongoing insurgency has added an extra US$4.5 billion to the costs. Terrorism has plagued the region since 2017, a few years after the discovery of its vast gas reserves.

Chief Executive Officer Patrick Pouyanné communicated the company’s position to Mozambique’s President Daniel Chapo in a letter dated 24 October 2025. TotalEnergies insists these costs be recognised as part of the investment, which would significantly reduce the project’s profits and the country’s tax income. The letter does not say how the US$4.5 billion was spent while the project was halted.

This could be the first of many bills related to the insurgency that Mozambique has to cover. The government has not yet reached an agreement with TotalEnergies regarding the additional costs; Chapo says negotiations between the two parties will now start.

What is clear is that gas exploration has become more expensive since the terrorism outbreak. Until the government finds a sustainable solution to the conflict, the risk of rising costs will remain high. Companies operating in conflict environments incur increased security expenses for their assets and staff, which are recorded as investment costs and are deductible from both revenues and taxes payable to the government.

Until a sustainable solution to the conflict is found, the risk of rising security costs will remain high

While there is no consensus on what exactly started the insurgency, the violence is linked to the discovery and beginning of gas exploration projects in Cabo Delgado. Several studies show that local communities did not benefit from job opportunities due to a lack of professional skills demanded by the projects.

At the same time, the government expropriated community land to allocate to gas companies. Communities’ access to the sea for fishing was also restricted, cutting off their primary sources of livelihood in what are predominantly rural and fishing areas. These factors contributed to the radicalisation of local populations, who later joined the terror groups.

Furthermore, instead of addressing the insurgency’s root causes and aggravating factors, the government deployed military and police contingents and hired foreign private military companies to counter the violence. The authorities should have addressed poverty, inequality, lack of access to the benefits of natural resources by local populations, and ethno-religious disputes, among other issues.

In many cases, residents were harassed or abused by the security forces, accused of collaborating with the insurgents, who themselves are mainly local. Rather than containing the instability, this approach added fuel to the fire, leading to the spread of intensified attacks to more areas.

After successive years of failed government responses, in March 2024 insurgents attacked the town of Palma, located about 10 km from the LNG site operated by TotalEnergies. The attack forced the declaration of force majeure and the suspension of the plant’s construction activities.

Two LNG sites, Cabo Delgado, Mozambique

 

The government is now a victim of its own decisions. When the conflict began in the resource-rich area, the state proceeded with gas exploration projects, creating mechanisms to protect corporate assets while disregarding the needs of nearby communities. This US$4.5 billion bill is the price Mozambique must pay for poor resource governance and for prioritising the protection of its resources over its people.

After TotalEnergies’ withdrawal, the government relied on foreign troops from Rwanda and the Southern African Development Community (SADC) to try to contain the violence. Still, it took almost five years for TotalEnergies to resume construction on the plant.

In addition to the steep extra costs, the company is demanding a 10-year extension of its exploration and production contract in the Rovuma Basin to compensate for the period of suspension.

Mozambique has shown little enthusiasm for the conditions imposed by TotalEnergies to restart the project, as they would entail significant revenue losses for the state. And extending the concession period by 10 years means Mozambique would have to wait much longer before the project is transferred to its ownership.

The gas exploration contract between the government and TotalEnergies follows a BOOT model – build, own, operate, transfer. This means that at the end of the concession period, the asset reverts to the state, or a new contract must be negotiated for an extension, subject to additional payments by the operator.

As long as the conflict endures, the projects will remain under threat – as will the state’s gas revenue

Although an agreement has yet to be reached between the parties, it appears likely that TotalEnergies will soon resume work on the project. However, violent conflict in the region persists. TotalEnergies is therefore expected to operate in a ‘green zone’ model – isolated from local communities – offering even fewer benefits to the surrounding population. This may fuel frustration among locals, further feeding the insurgency.

Another gas project, Rovuma LNG operated by ExxonMobil, is preparing to announce its final investment decision, estimated at around US$30 billion. Like the TotalEnergies project, this is an onshore venture that will likely face similar security challenges.

Although the insurgents currently show little capacity to directly attack the gas facilities, as long as the conflict endures, the projects will remain under threat – and so will government gas revenue.

To maximise gas gains for the state and extend their benefits to local communities, Mozambique’s government must adopt alternative conflict-resolution approaches beyond the military route, which has proven ineffective on its own. Investing in programmes that prevent and counter violent extremism and promote dialogue are key steps towards sustainable peace.

 

Exclusive rights to re-publish ISS Today articles have been given to Daily Maverick in South Africa and Premium Times in Nigeria. For media based outside South Africa and Nigeria that want to re-publish articles, or for queries about our re-publishing policy, email us.

Catastrophe bond investment funds in the UCITS format have continued to deliver strong returns for their investors through October, with the average 2025 year-to-date return for these cat bond funds running at 8.88% by the end of that month, according to the latest data from the Plenum CAT Bond UCITS Fund Indices.

Previously, the UCITS cat bond fund sector was running at an average return of 7.25% for the year to September 26th.

Since, then UCITS cat bond funds have on average added 1.51% in returns through the period to October 31st, a very strong few weeks of performance for the asset class.

Performance has been driven by coupons and spread developments in the catastrophe bond market, with some cat bond funds seeing near record monthly performance during the last two months.

Reviewing monthly performance for 2025 year-to-date, the Plenum CAT Bond UCITS Fund Indices delivered a 0.40% return for January, 0.32% for February, 0.56% for March, 0.28% for April, 0.52% for May, 0.58% for June, 1.09% for July, 1.34% for August and 1.38% for September up to the 26th.

Now, the latest data for the Plenum CAT Bond UCITS Fund Indices shows that from September 26th through to the end of October 2025 saw the average return across the group of UCITS catastrophe bond funds reach 1.51%, as the run-rate of returns continues to be strong.

The year-to-date average return of 8.88%, as of October 31st, demonstrates the continued attractiveness of the catastrophe bond asset class, while some funds have fared better and are nearing double-digits at this stage of the year, we understand.

For the full-year 2025, double-digits remains in sight for this index of UCITS catastrophe bond fund performance.

Without doubt, unless there is a major loss event then 2025 will be the third strongest year for the index in its history, by quite a margin.

For the period of September 26th through October 31st, lower-risk cat bond funds fared slightly better at 1.53%, while the higher-risk cat bond funds averaged a 1.49% return.

Year-to-date, higher risk UCITS cat bond funds averaged 8.94% while the lower-risk cohort averaged 8.90%.

On a rolling twelve month return basis, the average for the index stands at 11%, while for lower-risk cat bond funds it is 10.56% and higher-risk 11.42%.

These levels of performance remain very attractive historically for the catastrophe bond asset class and are more than adequate to continue driving investor interest.

Given the recent softening of pricing, across reinsurance in general and in the pricing of recent catastrophe bond issues though, it is hard to envisage double-digit returns being sustained on the rolling twelve-month basis for too much longer and that metric may revert to single digits in 2026 it currently seems.

Analyse UCITS cat bond fund performance, using the Plenum CAT Bond UCITS Fund Indices.

Analyse UCITS catastrophe bond fund assets under management using our charts here.

Analyse catastrophe bond market yields over time using this chart.