Innovations in Bacteriophage Genome Engineering for Combating Multidrug-Resistant Bacterial Infections.

High prevalence of multidrug resistant bacterial infections in Nepalese patients

Multidrug-resistant bacterial infections after liver transplantation: Prevalence, impact, and risk factors

Emergence of multidrug resistant infection in patients with severe acute pancreatitis

In recent years multi-drug resistant bacterial infections are on the rise. Various virulence factors play an important role in the pathogenesis of bacterial infections. One of them, which leads to recurrent and prolonged infection, is the ability of bacteria to form biofilms. Multiresistant and biofilm-associated infections are observed in patients with risk factors such as artificially implanted devices, chronic disease, immunosuppression, severe tissue structural damage due to trauma. The use of antibiotics in the treatment of these infections does not always ensure a successful outcome, so alternative treatments such as bacteriophage therapy are being evaluated and used. Bacteriophages or phages are bacterial viruses that are able to enter a specific bacterium, multiply in it and, at the end of their life cycle, lyse it. Phages are unaffected by bacterial resistance to antibiotics and have the potential to break down bacterial biofilms. Bacteria have the ability to acquire resistance to phages, therefore phage therapy is often combined with antibiotics. The interaction of these agents with planktonic and biofilm-forming cells is highly variable due to differences in the rates of interaction between phages and the bacterium, as well as the mechanisms of action of the antibiotics. The aim of this study is to evaluate the antimicrobial effect of bacteriophages and their interaction with antibiotics in vitro of selected multidrug-resistant and biofilm-forming bacteria and in the treatment of life-threatening infections. The dissertation is organised as a collection of four consecutive peer-reviewed articles. The structure of the study consists of three parts.In the first part investigation of the outcomes of S. aureus colonisation and biofilm formation in peritoneal dialysis patients over a two-year period was performed. Phenotypic differences in biofilm formation between different isolates of S. aureus, from the same individual were assessed. Bacteriophage lytic effect in isolated cultures was detected, and phage adaptation procedure to overcome bacteriophage resistance was performed. In the second part, the biofilm-forming capacity of uropathogenic E. coli isolates was investigated, the antibiotic and phage biofilm eradication capacity was determined. Bacteriophage adaptation was performed to overcome resistance and assess the lytic effect of adapted phages in planktonic and biofilm-forming cells. In the third part, the clinical efficacy of bacteriophages in patients with complicated multidrug-resistant P. aeruginosa osteomyelitis and Left Ventricular Assist Device (LVAD) associated infection was evaluated. Patients received local and systemic phage therapy supplemented with antibiotics and underwent wound revision. Clinical outcome and tolerability of therapy were analysed. This was preceded by bacteriophage selection, phage preparation and evaluation for clinical use. The effect of bacteriophages in biofilm models was also evaluated in the laboratory, the interaction of bacteriophages with the antibiotics used was determined and the development of phage resistance was assessed. The results show that biofilm-forming strains are common in the patient population studied and that their biofilm-forming capacity can vary widely. The biofilm-forming phenotype of the same bacterium may differ even within the same individual. The antibacterial effect of bacteriophages in the isolated bacteria was of broad lytic efficiency. In phage resistant S. aureus and E. coli isolates adaptation procedure led to overcoming phage resistance. After bacteriophage adaptation in E. coli culture, a strong biofilm eradication effect of the adapted bacteriophage was also observed. Treatment of patients with bacteriophages resulted in clinical recovery in both clinical cases. Complete eradication of P. aeruginosa was achieved in the patient with LVAD infection, whereas the patient with osteomyelitis had a relapse of infection after nine months in another bone segment, which was successfully treated with antibiotics. Different bacteriophages were used to treat the patients and their lytic and biofilm eradication effects were studied in combination with antibiotics. The bacteriophage BFC1.10 showed an additive effect with ceftazidime-avibactam, which rendered the causative organism sensitive to the antibiotic used. In contrast, phages PNM and PT07 had a better biofilm eradication effect when used first and then switched to ceftazidime-avibactam. Bacteriophage resistance to phages PNM and PT07 in P. aeruginosa culture developed as early as 12 h after the start of the experiment in the biofilm model. The development of resistance was different using different strains of the bacterium. The study shows that bacteriophages have a high potential to treat multidrug-resistant and biofilm-associated infections in both laboratory and human settings. Bacteriophage therapy can be complemented with antibiotics, but the interaction and sequence of administration of specific agents needs to be assessed as this has a significant impact on the efficacy of therapy. Although bacteriophage resistance is common in phage therapy, it can be overcome by using phage cocktails, an adaptation procedure or by combining phage therapy with antibiotics, even if the bacterium is moderately sensitive to the antibiotic. Further studies, especially clinical studies, are needed to evaluate phage therapy more precisely. 3.2 Clinical Medicine; Sub-Sector – Internal Medicine. Keywords: phage therapy, multidrug resistance, biofilm, phage resistance, phage adaptation, peritoneal dialysis, urinary tract infection, osteomyelitis, LVAD infection.

Multidrug-resistant (MDR) bacterial infections have become increasingly common in recent years due to the increased prevalence of diabetic foot ulcers (DFUs). We carried out a meta-analysis aimed at investigating the prevalence of MDR bacteria isolated from DFUs and analysing the risk factors for MDR bacterial infection in patients with DFUs. The PubMed/Medline, Web of Science, Embase, Cochrane Library, Ovid, Scopus, and ProQuest databases were searched for studies published up to November 2023 on the clinical outcomes of MDR bacteria in DFUs. The main outcome was the prevalence of MDR bacteria in DFUs. A total of 21 studies were included, representing 4885 patients from which 2633 MDR bacterial isolates were obtained. The prevalence of MDR bacteria in DFUs was 50.86% (95% confidence interval (CI): 41.92%-59.78%). The prevalence of MDR gram-positive bacteria (GPB) in DFUs was 19.81% (95% CI: 14.35%-25.91%), and the prevalence of MDR gram-negative bacteria (GNB) in DFUs was 32.84% (95% CI: 26.40%-39.62%). MDR Staphylococcus aureus (12.13% (95% CI: 8.79%-15.91%)) and MDR Enterococcus spp. (3.33% (95% CI: 1.92%-5.07%)) were the main MDR-GPB in DFUs. MDR Escherichia coli, MDR Pseudomonas aeruginosa, MDR Enterobacter spp., MDR Klebsiella pneumoniae, and MDR Proteus mirabilis were the main MDR-GNB in DFUs. The prevalence rates were 6.93% (95% CI: 5.15%-8.95%), 6.01% (95% CI: 4.03%-8.33%), 3.59% (95% CI: 0.42%-9.30%), 3.50% (95% CI: 2.31%-4.91%), and 3.27% (95% CI: 1.74%-5.21%), respectively. The clinical variables of diabetic foot ulcer patients infected with MDR bacteria and non-MDR bacteria in the included studies were analysed. The results showed that peripheral vascular disease, peripheral neuropathy, nephropathy, osteomyelitis, Wagner's grade, previous hospitalization and previous use of antibacterial drugs were significantly different between the MDR bacterial group and the non-MDR bacterial group. We concluded that there is a high prevalence of MDR bacterial infections in DFUs. The prevalence of MDR-GNB was greater than that of MDR-GPB in DFUs. MDR S. aureus was the main MDR-GPB in DFUs, and MDR E. coli was the main MDR-GNB in DFUs. Our study also indicated that peripheral vascular disease, peripheral neuropathy, nephropathy, osteomyelitis, Wagner's grade, previous hospitalization, and previous use of antibacterial drugs were associated with MDR bacterial infections in patients with DFUs.

Early clinical experience of bacteriophage therapy in 3 lung transplant recipients.

Pyelonephritis during pregnancy represents a significant maternal-fetal risk, particularly in the context of increasing multidrug-resistant (MDR) bacterial infections. This study aimed to characterize the microbiological profiles and antimicrobial resistance patterns of MDR pathogens causing pyelonephritis in pregnancy. Secondary objectives included the evaluation of patient characteristics, associated risk factors, and pregnancy outcomes. A retrospective comparative study was conducted including 171 pregnant patients hospitalized with acute pyelonephritis between 1 January 2017 and 30 April 2025. Thirty-four patients with MDR bacterial infections were compared with 137 patients with infections caused by pathogens with conserved antibiotic susceptibility (Non-MDR). Patients with MDR pyelonephritis were significantly older than those with Non-MDR infections (mean age 27.76 vs. 25.30 years, p = 0.03). MDR infections were more frequently diagnosed in the third trimester of pregnancy (58.8% vs. 29.9%, p = 0.003) and affected multiparous women more often (44.1% vs. 19.7%, p = 0.006). No statistically significant differences were observed between groups regarding clinical presentation or laboratory parameters (p > 0.05). Prior antibiotic exposure was significantly more common in the MDR group (85.29% vs. 26.61%, p < 0.001), as was a history of urological procedures, including urinary catheterization (29.41% vs. 15.10%, p = 0.009). For multivariate analysis, two factors were predictive for pyelonephritis with MDR pathogens: previous antibiotic treatment-OR 20.37 (95% CI 2.19-189.88) and urological procedures-OR 13.23 (95% CI 2.24-78-22). Escherichia coli was the predominant pathogen in both groups but was isolated more frequently in the Non-MDR cohort (81.75% vs. 58.82%, p = 0.015), followed by Klebsiella pneumoniae, which appeared more frequently in the study group (23.53% vs. 10.22%, p = 0.011). MDR isolates demonstrated significantly higher resistance rates to all tested antibiotics (p < 0.05). Complete resistance to ampicillin was observed in the MDR group (100%), compared with 58.01% in the Non-MDR group, indicating markedly reduced efficacy of this agent. Adverse neonatal outcomes were more frequent in the MDR group, with higher rates of Apgar scores < 7 at admission (23.5% vs. 8.8%, p = 0.01) and increased neonatal intensive care unit admission (20.6% vs. 7.3%, p = 0.02). For multivariate analysis, pyelonephritis with MDR pathogens was predictive for Neonatal Intensive Care Unit (NICU) admission (OR 8.17, 95% CI 2.41-27.67). These findings highlight the need for the periodic revision of empirical antibiotic protocols and risk-adapted therapeutic strategies in pregnant patients in order to reduce maternal and fetal morbidity.

Multidrug-resistant (MDR) bacterial infection is a common complication of severe acute pancreatitis (SAP). This study aimed to explore the association between human leukocyte antigen-antigen D-related (HLA-DR) expression and multidrug-resistant infection in patients with SAP. A total of 24 SAP patients who were admitted to Nanjing Drum Tower Hospital between May 2015 and December 2016 were enrolled in the study. The percentages of CD4+, CD8+, natural killer (NK), and HLA-DR (CD14+) cells and the CD4+/CD8+ cell ratio on days 1,7,14, and 28 after admission were determined by flow cytometry. Eighteen patients presented with the symptoms of infection. Among them, 55.6% patients (10/18) developed MDR infection. The most common causative MDR organisms were Enterobacter cloacae and Acinetobacter baumannii. The CD4+/CD8+ cell ratio and the percentage of NK cells were similar between patients with non-MDR and patients with MDR infections. In patients without infection, the HLA-DR percentage was maintained at a high level throughout the 28 days. Compared to the patients without any infection, the HLA-DR percentage in patients with non-MDR infection was reduced on day 1 but increased and reached similar levels on day 28. In patients with MDR infection, the HLA-DR percentage remained below normal levels at all-time points. It was concluded that persistent down-regulation of HLA-DR expression is associated with MDR bacterial infection in patients with SAP.

Background: There is an alarming increase in multi-drug resistant (MDR) bacterial infections worldwide. The Mediterranean region, including Jordan, reports a particularly high prevalence of hospital-acquired MDR infections, especially in Intensive Care Units (ICUs). Aim: To investigate the prevalence, antibiotic susceptibility, risk factors, and resistance genes associated with bacterial infections in ICUs across Jordan. Methodology: A total of 177 bacterial isolates were collected from 12 ICUs across Jordan between September 2019 and April 2022. Patient demographics, isolate characterization, antibiotic susceptibility, and resistance genes were recorded and analyzed. Results: The isolates included Klebsiella (22.6%), Staphylococcus (18.0%), Escherichia (17.5%), Acinetobacter (16.4%), Enterococcus (6.2%), Pseudomonas (6.2%), and others (13.0%). Of all isolates, 41.2% were MDR, 17.5% were extensive drug-resistant (XDR), and 6.8% were pandrug-resistant (PDR). MDR rates were highest in Klebsiella (82%), followed by Acinetobacter (79%), Pseudomonas (63%), Staphylococcus (56%), and Escherichia (51%). High resistance rates were observed for all antibiotics except vancomycin, colistin, linezolid, and teicoplanin (˂10%). Significant associations were found between MDR infections and Gram-negative bacteria, central lines, mechanical ventilation, nasogastric tubes, a history of recurrent infections, previous surgeries, bedridden patients, prophylactic antibiotic use, recent antibiotic exposure in the last two weeks, and elevated white blood cell counts (P˂0.05). MDR infections were also significantly linked to higher rates of complications and death (P˂0.05). The most common resistance genes identified were KPC in K. pneumoniae (28.6%), mecA for Staphylococci (62.5%), CTX-M among E. coli (48.4%), OXA-51 and OXA-23 genes in A. baumannii/spp (100%) and vanA gene for E. faecalis/spp (45.5%). Conclusion: ICUs serve as critical reservoirs for MDR bacterial infections. Implementation of nationwide, evidence-based antibiotic stewardship programs is strongly recommended.

Bacteriophages were discovered just over 100 years ago and have been used to treat bacterial infections in animals since the 1920s. The antimicrobial resistance crisis has led to a new surge of interest in the use of bacteriophage therapy as an alternative or supplement to antimicrobial therapy in humans and other animals. To describe the nature of bacteriophages and provide a critical review and update on the clinical use of bacteriophages in the treatment of challenging bacterial infections, with an emphasis on companion animal veterinary applications. The scientific literature on the subject was critically evaluated. Findings from the most pertinent papers have been presented in summary form and critiqued. Over the last 20 years there has been a considerable increase in the volume and quality of publications dealing with bacteriophage therapy. Some recent papers build on excellent work published in the 1980s and describe promising veterinary applications. Challenges related particularly to the registration and approval of phage remedies will need to be overcome before phage therapy can become a mainstream tool for use in veterinary settings. Considerably more research, particularly controlled clinical trials, needs to be done. Bacteriophage therapy is one of the most promising approaches to tackling the looming antimicrobial resistance crisis, yet substantial regulatory challenges will need to be overcome before it enters widespread use. Phage therapy also may, in the future, improve the management of challenging bacterial infections that are not necessarily multidrug-resistant.

Gram-negative bacterial infections, especially multidrug-resistant (MDR) bacterial infection, are becoming a serious threat to public health. Although it is widely accepted that both appropriate initial empirical therapy and targeted therapy are important, but for patients needing therapy adjustment, few studies have explored whether adjustment strategy based on microbiologic susceptibility test (MST) brings better outcome compared with empirical adjustment.A total of 320 patients with gram-negative bacterial infection (airway, blood, or pleural effusion) were selected and a prospective cohort study was conducted. Baseline characteristics and outcomes (microbiologic, clinical, and economic) were documented during follow-up.MDR and nosocomial infections were common among subjects. Initial therapies consistent with MST could result in reduced in-hospital mortality, treatment failure rate, infection-related death, percentages of patients needing therapy adjustment, and daily hospitalization cost with increased successful treatment rate compared with inconsistent with MST, and microbiologic outcomes were also better with appropriate therapies.For patients needing therapy adjustment, relying on MST gained no significant benefit on mortality, clinical, or microbiologic outcomes compared with depending on clinical experience. But for patients with MDR infection, adjustment relying on MST gained more benefit than non-MDR infection.Appropriate initial therapy significantly improved the prognosis of patients with gram-negative bacterial infections, but improvement was not that obvious for patients needing therapy adjustment which was based on MST compared with clinical experience, and more beneficial effects of adjustment relying on MST were obtained for patients with MDR bacterial infection.

Multidrug-resistant infection in COVID-19 patients: A meta-analysis

Patients in the neurosurgical intensive critical unit (ICU) face high rates of malnutrition and multidrug-resistant (MDR) infections. This study aimed to investigated the correlation between malnutrition and MDR bacterial infections, aiming to offer novel strategies for preventing and controlling MDR infections from the perspective of nutritional management in clinical practice. This retrospective cohort study analyzed 134 patients with MDR infections and 148 patients with non-MDR infections from November 2023 and May 2024 in neurosurgical ICU. MDR was defined as resistance to at least three antibiotic classes. Univariate, multivariate and correlation analyses were performed to explore the relationship between malnutrition and MDR infections. The incidence of malnutrition risk in the MDR group was significantly higher than in the non-MDR group (P < 0.05). After adjusting for confounding factors, malnutrition was still independently associated with an increased risk of MDR infections (OR = 2.336; 95% CI = 1.361~4.112, P = 0.002). Furthermore, C-reactive protein level was negatively correlated with TP (r = -0.281, P < 0.001), ALB (r = -0.267, P < 0.001), PLB (r = -0.279, P < 0.001), Hb (r = -0.167, P < 0.01) and PNI (r = -0.257, P < 0.001), suggesting that higher infection severity was associated with poorer nutritional status. For bacterial strains, Acinetobacter baumannii accounted for the largest proportion in our study. Malnutrition is an independent risk factor for MDR infection in neurosurgical ICU patients. This finding highlights the need for integrated interventions targeting malnutrition in MDR prevention for neurosurgical ICU patients.

Phage therapy is a promising treatment of multi-drug resistant (MDR) bacterial infections but is limited by the narrow host range of phage. To overcome this limitation, we developed a host range expansion (HRE) protocol that expands the host range of Pseudomonas aeruginosa-specific phage by cycles of co-incubation of phage with multiple P. aeruginosa strains. Application of the HRE protocol to a mixture of 4 phages, using 16 P. aeruginosa strains for development, resulted in undefined phage mixtures with greatly expanded host range. Individual phage clones derived from the undefined mixture had expanded host ranges but no individual clone could lyse all of the strains covered by the undefined mixture from which it was isolated. Reconstituting host range-characterized clones into cocktails produced defined cocktails with predictable and broad host ranges. The undefined mixture from the 30th cycle of the mixed-phage HRE (4ϕC30) showed a dose-dependent ability to prevent biofilm formation by, and to reduce a pre-existing biofilm of, 3 P. aeruginosa clinical isolates that produced high amounts of biofilm. A defined cocktail reconstituted from 3 host range-characterized clones had activity on high biofilm-formers susceptible to the phage. Phage therapy was superior to antibiotic therapy (levofloxacin) in a strain of P. aeruginosa that was resistant to levofloxacin. The HRE protocol establishes a rapid approach to create libraries of phage clones and phage cocktails with broad host range, defined composition and anti-biofilm activity.

Epidemiology of multi drug resistant gram negative bacteria in Kathmandu, Nepal