Trimethoprim Research Articles

Molecular Analysis of Dihydropteroate Synthase Gene Mutations in Pneumocystis jirovecii Isolates among Bulgarian Patients with Pneumocystis Pneumonia

Pneumocystis jirovecii pneumonia (PCP) is a significant cause of morbidity and mortality in immunocompromised people. The widespread use of trimethoprim-sulfamethoxazole (TMP-SMZ) for the treatment and prophylaxis of opportunistic infections (including PCP) has led to an increased selection of TMP-SMZ-resistant microorganisms. Sulfa/sulfone resistance has been demonstrated to result from specific point mutations in the DHPS gene. This study aims to investigate the presence of DHPS gene mutations among P. jirovecii isolates from Bulgarian patients with PCP. A total of 326 patients were examined via real-time PCR targeting the P. jirovecii mitochondrial large subunit rRNA gene and further at the DHPS locus. P. jirovecii DNA was detected in 50 (15.34%) specimens. A 370 bp DHPS locus fragment was successfully amplified in 21 samples from 19 PCP-positive patients, which was then purified, sequenced, and used for phylogenetic analysis. Based on the sequencing analysis, all (n = 21) P. jirovecii isolates showed DHPS genotype 1 (the wild type, with the nucleotide sequence ACA CGG CCT at codons 55, 56, and 57, respectively). In conclusion, infections caused by P. jirovecii mutants potentially resistant to sulfonamides are still rare events in Bulgaria. DHPS genotype 1 at codons 55 and 57 is the predominant P. jirovecii strain in the country.

Results of the Phase II of Epirchop Study, Evaluating the Efficacy of Tazemetostat in Combination with R-CHOP in Elderly Newly Diagnosed Diffuse Large B Cell Lymphoma (DLBCL): A Lysa Study

Introduction: Tazemetostat (TAZ) is a selective oral EZH2 inhibitor with a favorable safety profile and activity in pts with either EZH2wild type or mutant B-cell NHL. In the phase I part of Epi-RCHOP study, we reported that R-CHOP plus TAZ (Taz-RCHOP) was well tolerated with safety and PK results comparable to R-CHOP alone, and the RP2D of TAZ in combination with RCHOP was consistent with TAZ monotherapy (800mg BID). We report here the efficacy results of the phase II TAZ R-CHOP study (Epi-RCHOP, NCT02889523), in pts 60-80y with newly diagnosed DLBCL. Methods: Pts received 6 R-CHOP every 21 days in combination with continuous TAZ at 800 mg BID, plus 2 cycles of TAZ and R (cycle 7 and 8). Prophylaxis with G-CSF, valaciclovir and trimethoprim sulfamethoxazole were strongly recommended. Primary endpoint was PET-based complete metabolic response rate (CR, Lugano 2014) at end of study treatment (EOT, i.e 8 cycles or at time of permanent treatment discontinuation) (data cut-off was January 30, 2023). Sample size calculation was made with the expectation of an increase of 10% in CR, with an H0 hypothesis of 70% and an H1 assumption of 80%, leading to a theorical sample size of 122 pts, assuming a drop out of 5%. The efficacy set correspond to pts who signed an informed consent and received at least one dose of TAZ. Pts without response assessment (no matter the reason) were considered non-responders. Key secondary objectives were progression free and overall survival. A sensitivity analysis was performed excluding pts who withdrawn their consent (N=112). Analyses were conducted using SAS® version 9.3. Results: From July 31, 2020 to July 18, 2022, 122 pts were enrolled in 27 LYSA centers. Median age was 70 years (60-80), 57% were female, 12% had an ECOG-PS2+, 70.5% a stage IV disease and 19.7% stage III, 66% elevated LDH, 73.8% an IPI of 3-5. Mutational profile was available for 76 pts, of whom 10 (13%) had an EZH2 mutations and 26 (34%) and EZB profile as assessed with LymphGen classifier. One hundred pts (82%) received 8 cycles and 22 had a premature treatment discontinuation (PTD), 12 (9.8%) during the first 2 cycles and 10 (8.2%) between cycle 3 and 8. Reasons for PTD were: consent withdrawal (N=10, including 7 during cycle 1 & 2), adverse events (AE, N=6, including 2 treatment related), death (N=2, both due to septic shock), protocol deviation (N=2), progressive disease (N=1) or physician decision (N=1). Overall, the median percentage of TAZ dose received was 98.4% (mean 77.2%, Q1: 61%; Q3: 100%), and AE led to TAZ interruption in 4.1%, discontinuation in 2.8% and dose reduction in 2.4% of the cases. Mean time between cycles was 21.5 days. In oct 2020, after the inclusion of 22 pts, recommendation to cap vincristine at 1mg was done, due to an excess of constipation. At end of treatment or PTD, 92/122 pts (75.4%, IC95: 66.8-82.8%) achieved CR, 8 (6.6%) PR, 5 (4.1%) had a PD, 2 (1.6%) had died (septic shock) and 15 (12.3%) were not evaluated and considered as non-responders. The primary objective was not met in this efficacy set, with a CR of 75.4% (lower than the H1 at 80%). The sensitivity analysis, excluding the 10 pts that withdrawn their consent (all non-evaluated), showed a CR rate of 82.1% (92/112, IC95: 73.8-88.7%) and PR rate of 7.1%. After a median follow-up of 18.5 months (Range = [0.2; 28.1]), 17/122 pts had a relapse/progression (13.9%) and 12 had died: 4 due to lymphoma, 3 to COVID-19, 4 to AE (2 septic shock, 1 acute myeloid leukemia and 1 heart failure), 1 unknown cause (in CMR). The estimated 18m PFS and OS were 77.7% (IC95: 67.5-85.1%) and 88.8% (IC95: 79.9-93.9%) respectively. Most frequent AE were: neutropenia (53%), anemia (51%), nausea (47%), asthenia (42%), peripheral neuropathy (37%), gastro-intestinal hypomotility (35%), weight loss (28%), vomiting (24%), thrombocytopenia (24%). AE of grade 3 or more occurred in 73% of the pts and were mainly hematological AE with neutropenia (48%, including 6% of febrile neutropenia), anemia (23%), thrombocytopenia (17%) and gastro-intestinal disorder (overall 10.7%). Red blood cell and platelets transfusions were administered in 31% and 11.5% of the pts respectively. Conclusion: R-CHOP plus TAZ is doable. Efficacy results in this trial conducted during the COVID pandemic in a particularly challenging population, suggest that the combination warrant further investigation including correlative studies with molecular subclassification that will be presented.

Risk Factors and Prognostic Scoring Model for 28-Day Mortality in Hematologic Patients with Stenotrophomonas M altophilia Bacteremia

Objective: Patients with hematologic diseases are at a higher risk for Stenotrophomonasmaltophilia(SM) bacteremia, which poses significant burden on morbidity and mortality. The aim of this study was to evaluate the early predictors of 28-day mortality in hematologic patients with SM bacteremia and to construct a reliable prediction model which can be used to identify patients at higher risk for mortality. Methods: This retrospective analysis of 131 hospitalized patients diagnosed with SM bacteremia was conducted in a tertiary blood disease center in Tianjin, China, from January 2012 to June 2023. Univariate analysis and multivariate logistic regression analysis were used to identify prognostic risk factors and develop a prognostic mortality scoring system and internally validated by bootstrap method (resampling = 1000). Results: The overall 28-day mortality rate in our study was 29.77% (39/131). The mean age was 45 (IQR 30.5-56.0) years and 70 individuals (53.44%) were diagnosed with myeloid malignancies. The existing condition severity-related scores did not perform well in predicting 28-day mortality for the patients in our population (AUROC values: SOFA: 0.842; ECOG: 0.831; APACHE Ⅱ: 0.709; SAPS Ⅱ: 0.675; aCCI: 0.654; Pitt score: 0.629). Through univariate and multivariate analysis, we identified four independent risk factors associated with 28-day mortality: SOFA score (OR = 3.67, 95% CI 1.99-6.78, P < 0.001; score = corresponding SOFA score), age ≥ 60 years (OR = 6.95, 95% CI 1.44-33.55, P = 0.0158; score = 2), bloody sputum or hemoptysis (OR = 5.32, 95% CI 1.08-26.29, P = 0.0402; score = 2) and neutropenia duration ≥ 25 days (OR = 3.92, 95% CI 1.01-15.26, P = 0.0487; score = 1). These factors were used to develop a prognostic scoring model, which yielded good discrimination (AUC = 0.889, 95% CI: 0.829-0.949) and calibration (Hosmer-Lemeshow goodness-of-fit test P = 0.998), with similar performance in the internal bootstrap validation (AUC = 0.888). Furthermore, all patients were divided into the high-risk group (≥ 7 points) and the low-risk group (< 7 points) based on the model and compared using Kaplan-Meier curves and log-rank tests. Additionally, we founded that the use of trimethoprim-sulfamethoxazole (TMP/SMX) after infection was associated with a better prognosis ( P = 0.012). Conclusions: The prediction model, utilizing SOFA score, age, bloody sputum or hemoptysis and neutropenia duration, proves to be an effective tool for early identification of hematologic patients with SM bacteremia at high risk of mortality.

189. Outcomes of High-dose Oral Beta-lactam Step-down Therapy Compared to Alternative Oral Therapy for Bacteremic Urinary Tract Infections

Abstract Background Pyelonephritis and associated bacteremia are often treated with intravenous antibiotics followed by oral step-down therapy to a fluoroquinolone (FQ) or trimethoprim/sulfamethoxazole (TMP/SMX). Oral beta-lactams were historically utilized less often due to concern for treatment failure. Literature suggests oral beta-lactams are effective, but there are no preferred oral beta-lactam agents or dosing recommendations. This study evaluates outcomes of high-dose oral beta-lactam therapy for Enterobacterales bacteremia due to a urinary tract infection (UTI) compared to alternative therapy. Methods A retrospective, multicenter, observational cohort study was conducted evaluating patients admitted from February 1, 2020, to October 1, 2022, with gram-negative bacteremia from a urinary source. Adult patients were included if they received empiric intravenous antibiotic active against the bacteria isolated and transitioned to appropriately dosed oral cephalexin, amoxicillin, TMP/SMX, or FQ (Table 1). Excluded were patients who received less than 72 hours of oral therapy, had a diagnosis of renal abscess or lobar nephronia, and those who expired during admission. The primary outcome compared the composite of recurrent bacteremia or mortality within 30 days of completing oral therapy. Secondary outcomes included recurrent UTI, emergency department (ED) or hospital readmission, and Clostridioides difficile infections within 30 days of treatment completion. Results There were 194 patients who met inclusion criteria. The primary outcome occurred in 1/75 (1.3%) patients in the oral beta-lactam group and 2/119 (1.7%) patients in the FQ or TMP/SMX group (p=1.0). Two patients in the FQ or TMP/SMX expired within 30 days and 1 patient in the beta-lactam group had a recurrent bacteremia. There were 16 (21.3%) and 14 (11.8%) patients in the beta-lactam and FQ or TMP/SMX group, respectively, treated for recurrent UTI (p=0.073). Twenty (27%) patients in the beta-lactam group required ED re-evaluation or hospital readmission compared to 30 (25%) patients who received a FQ or TMP/SMX (p=0.74). Zero patients had a Clostridioides difficile infection. Conclusion Oral beta-lactams are as effective as oral FQ and TMP/SMX for the treatment of bacteremia from a urinary source. Disclosures All Authors: No reported disclosures

2738. Two-step sulfonamide antibiotic challenges in patients who require treatment with trimethoprim-sulfamethoxazole at an academic medical center: Outcomes in a largely immunocompromised population

Abstract Background Sulfonamide antibiotic allergy is the second most reported medication allergy. The allergy label limits the use of trimethoprim-sulfamethoxazole (TMP-SMX), the antibiotic of choice for pneumocystis jirovecii pneumonia (PJP), Toxoplasma, and many types of urinary tract infections. Immunocompromised patients, including solid organ transplant recipients, are considered to be at a higher risk for developing an infection. While penicillin allergies are commonly tested and patients delabeled, sulfonamide challenges are not as prevalent. Patients who are tested following a transplant may be on medications that lower the negative predictive value of the challenge, raising questions about whether they truly passed the challenge or whether mild reactions were masked. Methods: Thirty-seven patients labeled with a sulfonamide allergy who were evaluated and challenged with two doses of TMP-SMX were included. Adults (≥18 years) with a sulfonamide antibiotic allergy were evaluated and challenged between June 2020 and May 2023. Data collection included the number of patients challenged and delabeled, number of patients who had reactions and what type of reaction, and proportion of patients who completed the treatment or prophylaxis course for which TMP-SMX was the first-line antibiotic. Results Thirty-seven patients were challenged, of which twenty-seven patients were immunocompromised (73%). Thirty-four (92%) passed the sulfonamide challenge and were delabeled, and thirty-two (86%) completed the full course of TMP-SMX prophylaxis or treatment. Nineteen patients (51%) were tested following immunosuppression or antihistamine treatment due to a new transplant. Five patients were not able to be delabeled due to mild or delayed reactions to the challenge or treatment, including two patients that passed the sulfonamide challenge and were delabeled but had a mild reaction during SMX-TMP therapy and were relabeled. Conclusion The benefits of sulfonamide antibiotic allergy testing can have great effects in immunocompromised patients in preventing the use of second-line antibiotics. Sulfonamide challenges were shown to be safe and effective in immunocompromised patients in delabeling the sulfonamide antibiotic allergy and leading to the completion of treatment with TMP-SMX. Disclosures Shyam Joshi, MD, Cogent: Honoraria|Leo Pharma: Honoraria|Nectar Allergy: Advisor/Consultant|Nectar Allergy: Stocks/Bonds|Sanofi/Regeneron: Advisor/Consultant|Sanofi/Regeneron: Honoraria|Takeda: Honoraria

Outcomes of Children with Osteoarticular Infections Treated with Trimethoprim-Sulfamethoxazole versus Clindamycin

Abstract Background Early transition to oral antibiotic therapy for the treatment of children with osteoarticular infections (OAI; osteomyelitis [OM], septic arthritis [SA]) has become increasingly common, yet the choice of optimal regimen remains a challenge. With increasing resistance, poor palatability and reported allergies to commonly used oral antibiotics, the treatment options for children with OAI are limited. Trimethoprim-sulfamethoxazole (TMP-SMX) is a commonly used antibiotic, with activity against frequently encountered pathogens causing OAI, yet data regarding outcomes of children with OAI treated with TMP-SMX is limited. We sought to describe the characteristics and outcomes of children with OAI treated with TMP-SMX compared to clindamycin. Methods Records of children <18 years old, admitted to Riley Hospital for Children (Indianapolis, IN) between January 2010 to June 2021 for acute OAI were reviewed. Patients were identified by ICD-9/10 codes and an order for TMP-SMX or clindamycin. Patients were excluded if they did not receive TMP-SMX or clindamycin as their primary oral treatment of OAI, had symptoms >30 days, had a hardware associated infection, were immunocompromised or had an alternative diagnosis. Demographic, clinical, and outpatient/follow up data were recorded. Treatment was considered successful if the patient completed treatment with the discharge oral antibiotic (either TMP-SMX or clindamycin), and there was no evidence of infection at the end therapy. Treatment failure was defined as inability to tolerate the medication, development of an infection related complication, recurrent or chronic osteomyelitis. Additionally, significant adverse drug events (ADEs) were recorded. Results A total of 148 children met eligibility criteria (30 TMP-SMX and 118 clindamycin). Demographic and clinical characteristics are highlighted in Table 1. Children receiving TMP/SMX were younger (1.75 years) compared to those receiving clindamycin (7 years). Osteomyelitis was the most common infection type in both groups (47% in the TMP/SMX group vs. 48% in the clindamycin group). Staphylococcus aureus was the causative pathogen in 37% and 49%, while no organism was recovered in 57% and 43% in the TMP/SMX and clindamycin groups, respectively. Complications during hospitalization occurred in 13% in the TMP/SMX group, compared to 16% in the clindamycin group. The median dose of TMP-SMX was 13.4 mg/kg/day (IQR 11.3-14.9) and 38.7 mg/kg/day (IQR 33.4-40.2) for clindamycin. There was no difference in adverse drug events between the two groups with 2 patients (7%) in the TMP/SMX group developing an ADE compared to 13 patients (11%) in the clindamycin group (p=0.74, OR 0.58 [95% CI: 0.12-2.51]). Five (17%) of patients in the TMP/SMX group and 12 (10%) in the clindamycin group were lost to follow up. Of the patients that completed follow-up, there was no difference in treatment success with 84% (21 of 25) in the TMP/SMX and 92% (97 of 106) in the clindamycin group (p=0.27, OR 0.49 [95% CI: 0.15-1.54]) being successfully treated. Conclusions TMP-SMX was well tolerated and not associated with higher odds of treatment failure compared to clindamycin. A larger, prospective, randomized, comparative effectiveness study to control for potential confounding variables, and detect smaller, yet potentially clinically relevant outcome differences is warranted.

Ruthenium decorated V@WO3 nanocomposites heterostructures for selective detection of sulfonamide in honey samples

In the current study, V@WO3 nanocomposite heterostructures were synthesized by a facile hydrothermal method having 1D highly stable and uniform morphology. The designed nanostructures were decorated with ruthenium nanoparticles through a microwave-assisted fabrication method. The physiochemical attributes of Ru/V@WO3 were confirmed by applying various analytical as well as spectroscopic techniques; including, XRD, SEM, FTIR, Raman spectroscopy, and UV–vis spectroscopy. The Ru/V@WO3 modified glassy carbon electrode was utilized for the detection of sulfonamide. The fabricated electrode exhibited fantastic electrochemical properties for the highly selective and sensitive detection of sulfonamide with a desirable and well-defined reduction peak (39 µA) at − 0.6 V. The designed sensor was used for the detection of sulfonamide in honey samples with 0.006 nM LoD and the regression value was 0.997 indicating a good linear relationship between composite and analyte. To study the selectivity of the designed sensor, interfering substances; trimethoprim (TMP), metronidazole (MTD), cephalosporin (CEP), and dihydroartemisinin (DHA) were used and no response was recorded against these drugs. Furthermore, the modified GCE showed excellent stability and life span.

Acute septicemia and diagnostic evaluation of Aeromonas veronii infection in American bullfrogs (Aquarana catesbeiana)

In this epidemiological investigation of farmed bullfrogs spanning over 2 years, multiple batches of symptomatic American bullfrogs exhibiting red-leg syndrome and abdominal distension were identified across several farms. Subsequently, 12 type strains of Aeromonas veronii were isolated and identified from naturally infected bullfrogs. To assess the pathogenicity of these strains, 10 known virulence factors associated with high pathogenicity in A. veronii were examined. Among them, strain V10 possessed all 10 virulence genes and was selected as the key strain for subsequent artificial infection experiments. Infection assays on 30 bullfrogs, segmented by varying V10 strain doses (10 frogs/dose), indicated a survival of only 4, with 100% mortality at 1 × 108 CFU/mL. Additionally, all strains displayed resistance to vancomycin, sulfamethoxazole-trimethoprim (SMZ-TPM), lincomycin, penicillin, and ampicillin. Bacterial load assessment of blood and tissue samples indicated that the bacteria rapidly disseminated to the liver, spleen, and kidneys following host infection. Histopathological examinations indicated damage and inflammatory responses in the liver, spleen, kidney, and intestines. Furthermore, Hematological tests showed declines in white cells, hemoglobin, and platelets. Within 48 h post-infection, there was an upregulation of certain pro-inflammatory genes(C3, CXCL10, IL-1β, IL-8) and downregulation of anti-inflammatory(IL-4, IL-10) and antioxidant gene(GSH-px). These rerults indicate that infected bullfrogs can be diagnosed with acute septicemia. This is the first report on pathogenicity of A. veronii in bullfrogs, and it highlights its widespread prevalence and high resistance in farmed bullfrogs. These results have substantial implications for the prevention and treatment of A. veronii infections in aquaculture practices.

Trimethoprim inhibits renal H+-K+-ATPase in states of K+ depletion.

There is growing consensus that under physiological conditions, collecting duct H+ secretion is independent of epithelial Na+ channel (ENaC) activity. We have recently shown that the direct ENaC inhibitor benzamil acutely impairs H+ excretion by blocking renal H+-K+-ATPase. However, the question remains whether inhibition of ENaC per se causes alterations in renal H+ excretion. To revisit this question, we studied the effect of the antibiotic trimethoprim (TMP), which is well known to cause K+ retention by direct ENaC inhibition. The acute effect of TMP (5 µg/g body wt) was assessed in bladder-catheterized mice, allowing real-time measurement of urinary pH, electrolyte, and acid excretion. Dietary K+ depletion was used to increase renal H+-K+-ATPase activity. In addition, the effect of TMP was investigated in vitro using pig gastric H+-K+-ATPase-enriched membrane vesicles. TMP acutely increased natriuresis and decreased kaliuresis, confirming its ENaC-inhibiting property. Under control diet conditions, TMP had no effect on urinary pH or acid excretion. Interestingly, K+ depletion unmasked an acute urine alkalizing effect of TMP. This finding was corroborated by in vitro experiments showing that TMP inhibits H+-K+-ATPase activity, albeit at much higher concentrations than benzamil. In conclusion, under control diet conditions, TMP inhibited ENaC function without changing urinary H+ excretion. This finding further supports the hypothesis that the inhibition of ENaC per se does not impair H+ excretion in the collecting duct. Moreover, TMP-induced urinary alkalization in animals fed a low-K+ diet highlights the importance of renal H+-K+-ATPase-mediated H+ secretion in states of K+ depletion.NEW & NOTEWORTHY The antibiotic trimethoprim (TMP) often mediates K+ retention and metabolic acidosis. We suggest a revision of the underlying mechanism that causes metabolic acidosis. Our results indicate that TMP-induced metabolic acidosis is secondary to epithelial Na+ channel-dependent K+ retention. Under control dietary conditions, TMP does not per se inhibit collecting duct H+ secretion. These findings add further argument against a physiologically relevant voltage-dependent mechanism of collecting duct H+ excretion.

Microplastics aggravate the bioaccumulation and corresponding food safety risk of antibiotics in edible bivalves by constraining detoxification-related processes

Characterized by a sessile filter-feeding lifestyle, commercial marine bivalves inhabiting pollution-prone coastal areas may accumulate significant amounts of pollutants, such as antibiotic residues, in their soft tissues and thus pose a potential threat to the health of seafood consumers. Microplastics are another type of emerging pollutant that are prevalent in coastal areas and have been reported to interact with common antibiotics such as enrofloxacin (ENR) and trimethoprim (TMP). Nevertheless, little is known about the impacts of MPs on the accumulation and corresponding food safety risk of antibiotics in edible bivalve species. Taking the frequently detected ENR, TMP, and polystyrene (PS)-MPs as representatives, the accumulation of above-mentioned antibiotics in three commercial bivalves with or without the copresence of MPs was assessed. In addition, the corresponding food safety risks of consuming antibiotic-contaminated bivalves were evaluated. Moreover, the impacts of these pollutants on detoxification-related processes were analyzed using the thick-shell mussel as a representative. Our results demonstrated that blood clams (Tegillarca granosa), thick-shell mussels (Mytilus coruscus), and Asiatic hard clams (Meretrix meretrix) accumulated significantly higher amounts of antibiotics in their bodies under antibiotic-MP coexposure scenarios. Although based on the target hazard quotients (THQs) and the margins of exposure (MoEs) obtained, the direct toxic risks of consuming ENR- or TMP-contaminated bivalves were negligible, the TMP residue accumulated in TMP-MP-coexposed mussels did surpass the maximum residue limits (MRLs) of the corresponding National Food Safety Standard of China, suggesting that other forms of potential risks should not be ignored. In addition, it was shown that the detoxification, energy provision, and antioxidant capacities of the thick-shell mussels were significantly hampered by exposure to the pollutants. In general, our data indicate that MPs may aggravate the accumulation and corresponding food safety risk of antibiotics in edible bivalves by disrupting detoxification-related processes, which deserves closer attention.

Individual Keratinocyte Necroses in the Epidermis Are Apoptotic Keratinocytes in the Skin.

The patient was a 44-year-old woman with Stevens-Johnson syndrome due to receiving Baktar® (sulfamethoxazole trimethoprim) medication at our outpatient dermatology clinic. The epidermis, dermis, and subcutaneous adipose tissue samples showed numerous necrotic keratinocytes in the epidermis. Apoptotic nuclei were visualized as diaminobenzidine brown deposits with immunoperoxidase staining for cleaved caspase-3. The cleaved caspase-3-positive findings were consistent with eosinophilic material that appeared to be necrotic cells within the epidermis. Therefore, these eosinophilic materials may be apoptotic bodies. Generally speaking, eosinophilic cells are considered necrotic keratinocytes, especially in Japan. To the best of our knowledge, no studies have used apoptotic immunohistochemical markers to examine whether these structures are apoptotic in a human specimen.

The environmental risk assessment of constructed wetlands filled with iron and manganese ores in typical antibiotic treatment

Constructed wetlands (CWs) is considered as an efficient and environmentally friendly technology for advanced wastewater treatment to eliminate organic pollutants such as sulfamethoxazole (SMX) and trimethoprim (TMP). Iron (Fe) and manganese (Mn) ores have attracted more and more attention as CWs substrates in treating SMX and TMP, but the potentially negative environmental effects of wetland effluents, ore contaminants leached from the substrates and the risk of transmission of antibiotic resistance genes (ARGs) are still not clear. Three CW groups with different substrates (river sand (C-CW), Fe ore (Fe-CW), and Mn ore (Mn-CW)) were set up to evaluate the average removal rates and environmental risk in treating wastewater containing SMX and TMP. The results showed that the average removal rates of SMX and TMP by Fe-CW and Mn-CW were significantly higher than that of C-CW by 12.46%, 6.59% and 38.93%,15.39% respectively (p < 0.05), suggesting that both Fe and Mn ores facilitated the removal of antibiotics. However, the least abundance of ARGs was found in the layer of Fe ore at the middle layer (ML) in Fe-CW among all CWs, which suggested that Fe ore could reduce the risk of ARGs transmission. Although the environmental risk of Fe-CW and Mn-CW effluent was low, Fe-CW effluent inhibited the growth of Chlorella in both 48h and 72h experiments, while Mn-CW effluent showed an inhibitory effect in 48h and then promoted the growth in 72h. Meanwhile, these findings offer valuable insights for wetland health assessment and substrate selection for CWs.

Regulation of eDHFR-tagged proteins with trimethoprim PROTACs

Temporal control of protein levels in cells and living animals can be used to improve our understanding of protein function. In addition, control of engineered proteins could be used in therapeutic applications. PRoteolysis-TArgeting Chimeras (PROTACs) have emerged as a small-molecule-driven strategy to achieve rapid, post-translational regulation of protein abundance via recruitment of an E3 ligase to the target protein of interest. Here, we develop several PROTAC molecules by covalently linking the antibiotic trimethoprim (TMP) to pomalidomide, a ligand for the E3 ligase, Cereblon. These molecules induce degradation of proteins of interest (POIs) genetically fused to a small protein domain, E. coli dihydrofolate reductase (eDHFR), the molecular target of TMP. We show that various eDHFR-tagged proteins can be robustly degraded to 95% of maximum expression with PROTAC molecule 7c. Moreover, TMP-based PROTACs minimally affect the expression of immunomodulatory imide drug (IMiD)-sensitive neosubstrates using proteomic and biochemical assays. Finally, we show multiplexed regulation with another known degron-PROTAC pair, as well as reversible protein regulation in a rodent model of metastatic cancer, demonstrating the formidable strength of this system. Altogether, TMP PROTACs are a robust approach for selective and reversible degradation of eDHFR-tagged proteins in vitro and in vivo.

Trends in the susceptibility of U.S. Acinetobacter baumannii-calcoaceticus species complex and Stenotrophomonas maltophilia isolates to minocycline, 2014-2021.

Acinetobacter baumannii-calcoaceticus species complex (ACB) and Stenotrophomonas maltophilia (SM) are opportunistic, non-fermentative organisms that can cause serious hospital-acquired infections in immunocompromised patients. These pathogens are inherently resistant to several common drug classes and often acquire other resistance mechanisms, making them difficult to treat. In this study, we analyzed the susceptibility of 1,029 contemporary ACB and 1,522 SM isolates to minocycline (MIN) and levofloxacin (LEV) as well as meropenem (MER) for ACB and trimethoprim-sulfamethoxazole (TMP/SMX) for SM using the CLSI broth microdilution method. Isolates were collected as a part of the SENTRY Antimicrobial Surveillance Program from 2014 to 2021. Pneumonia in hospitalized patients was the most common infection from which ACB (57.0%) and SM (73.9%) were isolated. MIN had the highest in vitro activity for ACB (86.2%) and SM (99.5%). The activity of ACB and SM to all three agents varied over the period studied. MIN activity to ACB decreased in 2020 (80.6%) but rebounded in 2021 (86.2%). LEV and MER showed an overall trend of increasing susceptibility for ACB, with slightly lower activity in 2020-2021. MIN and TMP/SMX (>98.3% and >93.7%, respectively) activities were stable against SM isolates. LEV activity decreased from 84.3% (2015) to 69.2% (2018). The activity of MIN remained stable and higher than other agents tested for both ACB and SM, pathogens that have limited therapeutic alternatives. These in vitro data suggest that MIN is a useful treatment option for infections caused by ACB or SM.IMPORTANCEAcinetobacter baumannii-calcoaceticus species complex and Stenotrophomonas maltophilia are opportunistic, non-fermentative Gram-negative organisms that can cause serious hospital-acquired infections in immunocompromised patients. These pathogens are inherently resistant to several common drug classes and often acquire other resistance mechanisms, making them difficult to treat. In this study, we analyzed the trends of susceptibility of over 2,500 U.S. bacterial isolates collected from hospitalized patients over an 8-year period to minocycline, which is used to treat infections caused by these pathogens. These in vitro data suggest that minocycline is a useful treatment option for infections caused by Acinetobacter baumannii-calcoaceticus species complex or Stenotrophomonas maltophilia.

A genetically encoded protein tag for control and quantitative imaging of CAR T cell therapy

Chimeric antigen receptor (CAR) Tcell therapy has been successful for hematological malignancies. Still, a lack of efficacy and potential toxicities have slowed its application for other indications. Furthermore, CAR Tcells undergo dynamic expansion and contraction invivo that cannot be easily predicted or controlled. Therefore, the safety and utility of such therapies could be enhanced by engineered mechanisms that engender reversible control and quantitative monitoring. Here, we use a genetic tag based on the enzyme Escherichia coli dihydrofolate reductase (eDHFR), and derivatives of trimethoprim (TMP) to modulate and monitor CAR expression and Tcell activity. We fused eDHFR to the CAR C terminus, allowing regulation with TMP-based proteolysis-targeting chimeric small molecules (PROTACs). Fusion of eDHFR to the CAR does not interfere with cell signaling or its cytotoxic function, and the addition of TMP-based PROTACs results in a reversible and dose-dependent inhibition of CAR activity via the proteosome. We show the regulation of CAR expression invivo and demonstrate imaging of the cells with TMP radiotracers. Invitro immunogenicity assays using primary human immune cells and overlapping peptide fragments of eDHFR showed no memory immune repertoire for eDHFR. Overall, this translationally-orientied approach allows for temporal monitoring and image-guided control of cell-based therapies.

Incidence and Outcomes of Pneumocystis Pneumonia Following Chimeric Antigen Receptor T-Cell Therapy: A Real-World Analysis

Background: Pneumocystis Pneumonia (PCP) is a known opportunistic infection after chimeric antigen receptor (CAR) T-cell therapy. However, the incidence and outcomes of PCP are poorly understood, and there has been no clear consensus on the optimal duration for PCP prophylaxis post-CAR T-cell treatment. Methods: From a large real-world database, TriNetX Research Network, we identified patients who underwent commercial anti-CD19 and anti-BCMA CAR T-cell therapy between January 2019 and January 2023. The diagnosis of PCP was established using the ICD-10-CM code B59 (pneumocystosis) or through Logical Observation Identifiers Names and Codes (LOINC) documentation of positive microbiologic tests from respiratory samples. Results: Among the 1,107 and 280 patients who received anti-CD19 and anti-BCMA CAR T-cell therapies, 19 (1.7%) and 4 (1.4%) patients developed PCP, respectively. Altogether, a total of 23 (1.7%) patients developed PCP within a median of 139 days (range 19 to 732 days) post-infusion. (Figure) Demographics, the incidence and severity of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), and the initial choice for PCP prophylaxis, were similar for patients with and without PCP. Patients who developed PCP were more likely to have used dexamethasone (65 vs 43%, p=0.02) and have a shorter duration of trimethoprim/sulfamethoxazole (TMP/SMX) prophylaxis (median 9 [range 1 to 45] vs 19 [range 1 to 106] weeks, p=0.002). In the univariate analysis, patients who received TMP/SMX prophylaxis for less than 3 months showed a trend of a higher incidence of PCP compared to those who received it for more than 3 months (hazard ratio [HR] 3.54, 95% confidence interval [CI] 0.87 to 14.3, p=0.06). (Table) Regarding survival outcomes, patients with and without PCP had similar time-to-next-treatment or death (median 84 vs 68 days, HR 0.96, 95%CI 0.61 to 1.51) and overall survival (median 518 days vs not-reached, HR 1.61, 95%CI 0.91 to 2.88). Conclusions: In real-world conditions with routine prophylaxis, PCP is an infrequent opportunistic infection following anti-CD19 or anti-BCMA CAR T-cell therapy. Patients with and without PCP were associated with similar survival outcomes.

Sub-therapeutic trimethoprim and sulfamethoxazole plasma concentrations during continuous venovenous hemofiltration in a patient with COVID-19 and pulmonary Pneumocystis jirovecii co-infection: Acase report.

To investigate drug concentration of trimethoprim-sulfamethoxazole (TMP-SMX) using therapeutic drug monitoring (TDM) for severe Pneumocystis jirovecii (PJP) infection in a critically ill patient with COVID-19 receiving continuous venovenous hemofiltration treatment and regional citrate anticoagulation (RCA-CCVH). A 72-year-old man with hypoxemic respiratory failure due to COVID-19 infection was admitted to the intensive care unit for invasive mechanical ventilation. The patient developed acute renal failure that required RCA-CVVH. Pulmonary co-infection with PJP was diagnosed, and a high TMP-SMX dose was initiated according to (inter)national guidelines with dose reduction after 3 days because of renal failure. Population pharmacokinetics were assessed for TMP and SMX as well as clearance by RCA-CVVH, volume of distribution, and time above threshold levels for measured plasma concentrations. During renal failure requiring RCA-CVVH, a corresponding dose reduction of TMP-SMX to 320/1,600 mg twice a day, according to current Dutch SWAB and Dutch Association of Hospital Pharmacists guidelines, resulted in unintended under-dosing with sub-therapeutic TMP-SMX concentrations. Pharmacokinetic modeling and dose adjustment of TMP-SMX to 640/3,200 mg 3 times daily resulted in steady-state TMP-SMX peak concentrations associated with efficacy against PJP. Hence, the patient was successfully weaned from the ventilator and discharged. We hypothesize that our new dose recommendation of 640/3,200 mg TMP-SMX 3 times daily is associated with an increased probability of critical patients being successfully liberated from mechanical weaning following PJP pneumonia and COVID-19 infection.

Prevalence and genomic characterization of Salmonella isolates from commercial chicken eggs retailed in traditional markets in Ghana.

Salmonella enterica are important foodborne bacterial pathogens globally associated with poultry. Exposure to Salmonella-contaminated eggs and egg-related products is a major risk for human salmonellosis. Presently, there is a huge data gap regarding the prevalence and circulating serovars of Salmonella in chicken eggs sold in Ghana. In this study, 2,304 eggs (pools of six per sample unit) collected from informal markets in Accra, Kumasi and Tamale, representing the three ecological belts across Ghana, were tested for Salmonella. Antimicrobial susceptibility testing and Whole Genome Sequencing (WGS) of the isolates were performed using standard microdilution protocols and the Illumina NextSeq platform, respectively. The total prevalence of Salmonella was 5.5% with a higher rate of contamination in eggshell (4.9%) over egg content (1.8%). The serovars identified were S. Ajiobo (n = 1), S. Chester (n = 6), S. Hader (n = 7), S. enteritidis (n = 2); and S. I 4:b:- (n = 8). WGS analysis revealed varied sequence types (STs) that were serovar specific. The S. I 4:b:- isolates had a novel ST (ST8938), suggesting a local origin. The two S. enteritidis isolates belonged to ST11 and were identified with an invasive lineage of a global epidemic clade. All isolates were susceptible to ampicillin, azithromycin, cefotaxime, ceftazidime, gentamicin, meropenem, and tigecycline. The phenotypic resistance profiles to seven antimicrobials: chloramphenicol (13%), ciprofloxacin (94%), and nalidixic acid (94%), colistin (13%), trimethoprim (50%) sulfamethoxazole (50%) and tetracycline (50%) corresponded with the presence of antimicrobial resistance (AMR) determinants including quinolones (gyrA (D87N), qnrB81), aminoglycosides (aadA1), (aph(3")-Ib aph(6)-Id), tetracyclines (tet(A)), phenicols (catA1), trimethoprim (dfrA14 and dfrA1). The S. enteritidis and S. Chester isolates were multidrug resistant (MDR). Several virulence factors were identified, notably cytolethal distending toxin (cdtB gene), rck, pef and spv that may promote host invasion and disease progression in humans. The findings from this study indicate the presence of multidrug resistant and virulent strains of Salmonella serovars in Ghanaian chicken eggs, with the potential to cause human infections. This is a critical baseline information that could be used for Salmonella risk assessment in the egg food chain to mitigate potential future outbreaks.

Prevalence of multidrug resistance and extended-spectrum beta-lactamase-producing Klebsiella pneumoniae from dairy cattle farm wastewater in East Java Province, Indonesia

Background and Aim: Antibiotic resistance in Klebsiella pneumoniae, especially extended-spectrum beta-lactamase (ESBL) producers, has become a global public health problem. This study aimed to estimate the prevalence of multidrug resistance (MDR) and ESBL-producing K. pneumoniae in wastewater from dairy farms. Materials and Methods: This study was conducted on dairy farms in East Java Province from June to October 2022. In total, 342 dairy farm wastewater samples were isolated on buffered peptone water media from six cities/regencies with the highest dairy cattle population in East Java. Samples were identified using MacConkey agar media, Gram-staining,eosin-methylene blue agar, and biochemical tests. In total, 14.32% (49/342) samples contained K. pneumoniae. Positive isolates were tested for antibiotic sensitivity. Klebsiella pneumoniae resistant to beta-lactam was confirmed using the double-disk synergy test to confirm the presence of ESBL-producing bacteria. Results: The percentage of antibiotic resistance in K. pneumoniae was 98% resistance to ampicillin, 67.3% to cefotaxime, 46.9% to tetracycline, 49% to ciprofloxacin, 98% to streptomycin, 14.3% to sulfamethoxazole–trimethoprim, and 83.7% to chloramphenicol. The prevalence of MDR in K. pneumoniae was 12.57% (43/342), with the highest prevalence in the five classes of antibiotics at 41.86% (18/43), and the prevalence of ESBL-producing K. pneumoniae was 5.55% (19/342), with the highest prevalence in the districts of Blitar and Pasuruan at 26.31% (5/19). Conclusion: Although the prevalence of ESBL-producing K. pneumoniae in wastewater samples from dairy farms was low, caution is recommended because they can be a reservoir for ESBL. Keywords: animal health, dairy cattle, extended-spectrum beta-lactamase, human health, Klebsiella pneumoniae, wastewater.

Construction and functional verification of size-reduced plasmids based on TMP resistance gene dfrB10.

Plasmid size is one of the factors affecting transfection efficacy in most of the molecular genetic research studies. One effective approach for reducing plasmid size is to replace relatively large, conventional antibiotic resistance genes with the short-size dfrB10 gene. The successful construct of a series of dfrB10-based tool plasmids and their functional validation, via comparison with original plasmids, suggest that dfrB10 is a potent drug resistance selection marker. The antibiotic trimethoprim offers convenient usage comparable to that of ampicillin or kanamycin. Additionally, fluorescence analysis has demonstrated the compatibility of TMP with protein expression in various host cells. Based on these findings, TMP-dfrB10 could be an alternative choice for future use in molecular genetic research studies that require miniature plasmids to achieve optimal results.