Opportunistic Infections Research Articles

Positron Emission Tomography Imaging of Acinetobacter baumannii Infection: Comparison of Gallium-68 Labeled Siderophores.

Acinetobacter baumannii (AB) is an opportunistic pathogen with growing clinical relevance due to its increasing level of antimicrobial resistance in the last few decades. In the event of an AB hospital outbreak, fast detection and localization of the pathogen is crucial, to prevent its further spread. However, contemporary diagnostic tools do not always meet the requirements for rapid and accurate diagnosis. For this reason, we report here the possibility of using gallium-68 labeled siderophores, bacterial iron chelators, for positron emission tomography imaging of AB infections. In our study, we radiolabeled several siderophores and tested their in vitro uptake in AB cultures. Based on the results and the in vitro properties of studied siderophores, we selected two of them for further in vivo testing in infectious models. Both selected siderophores, ferrioxamine E and ferrirubin, showed promising in vitro characteristics. In vivo, we observed rapid pharmacokinetics and no excessive accumulation in organs other than the excretory organs in normal mice. We demonstrated that the radiolabeled siderophores accumulate in AB-infected tissue in three animal models: a murine model of myositis, a murine model of dorsal wound infection and a rat model of pneumonia. These results suggest that both siderophores radiolabeled with Ga-68 could be used for PET imaging of AB infection.

T-cell activation and senescence in asymptomatic HIV/Leishmania infantum co-infection.

Leishmania infantum can be an opportunistic pathogen, with an immunocompromised status increasing the risk of converting asymptomatic infection to symptomatic visceral leishmaniasis (VL). VL has approximately 5% fatality rate; and HIV coinfection (AIDS/VL) increases this risk. We hypothesized that, relative to those with HIV alone, people with co-infection would have altered T cell activation which could impact on the risk of VL. A cross-sectional study was performed between 2014 and 2016 to determine the prevalence of L. infantum infection in people living with HIV (PLHIV) residing in Brazil (n = 1,372). Subsequent incident cases of VL were ascertained from a public health database through 2018 and from a cohort of families with VL. Immune status of 69 participants was evaluated and comparisons made between those with and without HIV, with latent or with active Leishmania infection and those without HIV but with active or resolved Leishmania or T cell hypersensitivity to Leishmania antigen and healthy control subjects. A total of 24.2% of PLHIV had positive anti-IgG L. infantum antibodies. The relative risk of developing AIDS/VL was 2.27 (95% CI: 0.920 to 5.59; p = 0.07) to HIV/Leish coinfected subjects with positive leishmania serology compared to HIV subjects without leishmania serology. Poor adherence to antiretroviral therapy (p = 0.0008) or prior opportunistic infections (p = 0.0007) was associated with development of AIDS/VL in asymptomatic HIV/Leish. CD4+ and CD8+ T cells counts or viral load were similar between asymptomatic HIV/Leish and HIV subjects. However, activated CD8+CD38+HLA-DR+ T cells were higher in asymptomatic HIV/Leish than HIV. Likewise, senescent (CD57+) and PD1+ CD8+ T cells were higher in asymptomatic HIV/Leish than in AIDS/VL or HIV groups. Although asymptomatic HIV/Leish subjects had CD4+ and CD8+ T cells similar to HIV alone, their CD8+T cells had increased activation and senescence which could contribute to risk of developing VL.

Identification of Antibacterial Agents with a Synergistic Effect in Pteris vittata L. by Exploration of Metabolome Data and Multivariate Analysis.

In metalliferous ecosystems, bacteria could develop membrane efflux pumps to extrude heavy metals. Native plants, frequently exposed to such bacteria, may serve as rich chemical reservoirs for discovering potential antibacterial and/or resistance-modifying agents. This study aims to investigate the antibacterial properties of Pteris vittata L., a metal hyperaccumulator abundant in metal-rich environments, against Stenotrophomonas maltophilia K279a, an opportunistic nosocomial pathogen linked to severe respiratory tract infections. A novel approach was used, employing the metabolome of P. vittata in a partial least squares regression model to predict the correlation between the whole chemical profile and activity. Marked compounds were then subjected to invitro test for bactericidal and synergistic properties using broth dilution and time-kill assays. The multivariate analysis demonstrated a strong synergistic effect among a range of flavonoids compared to the individual responses. Notably, nine flavonoids were confirmed to reduce the minimum inhibitory concentration and minimal bactericidal concentration values of active compounds, cyanidin-3,5-O-diglucoside and perlagonin, from 64.0 and 108 μg/mL to 0.25 and 3.38 μg/mL, respectively. These results provide insights into the possibility of combating clinical infections caused by S. maltophilia and highlight the potential of exploring new synergetic interactions among natural products as an alternative to the conventional bio-guided isolation strategy.

Adiponectin pathway activation dampens inflammation and enhances alveolar macrophage fungal killing via LC3-associated phagocytosis.

Although innate immunity is critical for antifungal host defense against the human opportunistic fungal pathogen Aspergillus fumigatus, potentially damaging inflammation must be controlled. Adiponectin (APN) is an adipokine produced mainly in adipose tissue that exerts anti-inflammatory effects in adipose-distal tissues such as the lung. We observed increased mortality and increased fungal burden and inflammation in neutropenic mice with invasive aspergillosis (IA) that lack APN or the APN receptors AdipoR1 or AdipoR2. Alveolar macrophages (AMs), early immune sentinels that detect and respond to lung infection, express both receptors, and APN-deficient AMs exhibited an inflammatory phenotype that was associated with decreased fungal killing. Pharmacological stimulation of AMs with AdipoR agonist AdipoRon rescued deficient killing in APN-/- AMs and was dependent on the presence of either receptor. Finally, APN-enhanced fungal killing was associated with increased activation of the non-canonical LC3 pathway of autophagy. Thus, our study identifies a novel role for APN in LC3-mediated killing of A.fumigatus.

Cronobacter Surveillance in Minnesota, United States, 2002-2024.

Cronobacter sakazakii is an opportunistic pathogen associated with bloodstream infections and meningitis in neonates and infants. C. sakazakii isolated from infants under 1 year of age has been reportable in Minnesota since 2003. Invasive Cronobacter infections in infants was recently made notifiable nationally in 2024. Therefore, this summary of over 20 years of reportable surveillance for C. sakazakii from Minnesota infants will inform other jurisdictions. During 2002-2024, 23 C. sakazakii cases in infants, including 8 invasive and 15 non-invasive cases, were identified and confirmed using MALDI-TOF or conventional biochemical methods. Invasive cases were younger (median, 18 days; range, 8 days to 10 months) than non-invasive cases (median, 98 days; range, 7 days to 8 months). Cronobacter isolates from infant (21), child (4), and adult (15) cases were submitted for whole genome sequencing (WGS) to obtain a contextual understanding of potential relatedness. In addition, C. sakazakii isolated from powdered infant formula (PIF) in 2003 was sequenced for comparison. Sequence type (ST) 4 was responsible for the majority of C. sakazakii cases overall (65%) and invasive infant cases (88%). Opened and unopened PIF was cultured from 10 investigations representing both invasive and non-invasive cases. Two (25%) opened PIF samples tested positive for C. sakazakii ST 4 while one (11%) unopened PIF sample tested positive for C. sakazakii ST 4. WGS demonstrated that no clinical cases were associated with a recognized outbreak. Several cases were likely due to contamination of PIF in the home during preparation, highlighting the importance of infant caregiver education to prevent infections.

Polystyrene Microplastics Can Aggravate the Damage of the Intestinal Microenvironment Caused by Okadaic Acid: A Prevalent Algal Toxin

As emerging contaminants, microplastics (MPs) may pose a threat to human health. Their co-exposure with the widespread phycotoxin okadaic acid (OA), a marine toxin known to cause gastrointestinal toxicity, may exacerbate health risk and raise public safety concern. In this study, the toxicity mechanisms of MPs and OA on intestinal microenvironment was explored using human Caco-2 cells as the model, which was combined with an in vitro fecal fermentation experiment. Our results showed that co-exposure to MPs (80 μg/mL) and OA (20 ng/mL) significantly decreased cell viability, increased intracellular reactive oxygen species (ROS) production, elevated lactate dehydrogenase release, impaired ABC transporter activity, promoted OA accumulation, and triggered inflammatory response compared to the control, MPs, and OA groups, indicating that co-exposure directly compromises intestinal epithelial integrity. In vitro fermentation experiments revealed that co-exposure disrupted gut microbial composition, decreasing the relative abundance of some bacteria, such as Parasutterella and Adlercreutzia, while increasing opportunistic pathogens, such as Escherichia-Shigella, increased. These findings provide new insights into the impact and underlying mechanisms of MPs and OA co-exposure on intestinal homeostasis, highlighting the potential health risks associated with MPs.

Age matters: exploring differential effects of antimicrobial treatment on gut microbiota of adult and juvenile brown trout (Salmo trutta)

BackgroundAntibiotics and antiparasitics are essential tools in controlling infectious disease outbreaks in commercial aquaculture. While the negative effects of antimicrobials on the gut microbiome of various farmed fish species are well documented, the influence of underlying host factors, such as age, on microbiome responses remains poorly understood. This is especially evident for peracetic acid, whose impact on the gut microbiome has not yet been studied. Understanding how microbiome dynamics vary by host age is critical to improving antibiotic stewardship in aquaculture. In this study, juvenile and sexually mature brown trout (Salmo trutta) were used as a model to investigate the age-dependent effects of florfenicol and peracetic acid on the gut microbiome using a 16S rRNA metabarcoding approach.ResultsFish age significantly shaped taxonomic composition and microbial co-occurrence network structure of the gut microbiome, regardless of treatment. Juvenile trout exhibited greater microbiome volatility and a stronger response to both florfenicol and peracetic acid compared to adult fish, with disruptions persisting up to 11 days post-treatment. Temporal dynamics were also evident, with microbial shifts characterized by a decline in beneficial commensals like Cetobacterium and Lactococcus. Although overall abundance recovered by 18 days post-treatment, network positions of key microbial community members remained altered, particularly in juvenile fish. Opportunistic pathogens, including Aeromonas and Streptococcus, were enriched and assumed more central roles within the microbial networks in treated fish.ConclusionThe initial composition of the gut microbiome in brown trout is strongly influenced by fish age, which in turn affects the microbiome’s response to antibiotic disruption. Juveniles displayed higher susceptibility to microbiome perturbation, and although recovery was observed at the community level, network properties remained altered. This study also provides the first evidence that external peracetic acid application can disrupt gut microbial communities. Since compositional shifts are often linked to functional alterations, even short-term disruptions may have important consequences for host health in developing fish. These findings emphasize the importance of considering gut microbial community structure in relation to fish age in aquaculture management practices.

Genomic Characterization of Extremely Antibiotic-Resistant Strains of Pseudomonas aeruginosa Isolated from Patients of a Clinic in Sincelejo, Colombia

Pseudomonas aeruginosa is an opportunistic pathogen classified as a priority and a great public health concern; therefore, this research focuses on the genomic characterization of extremely resistant strains of P. aeruginosa isolated from patients in a clinic in Sincelejo, Colombia. Seven strains were analyzed by whole genome sequencing using the Illumina NovaSeq platform, with a focus on the identification of resistance genes and virulence factors through the CARD and VFDB databases. An ANI (Average Nucleotide Identity) analysis was carried out to determine the genetic relationship between the strains, complemented by a phylogenomic analysis to place the strains in different evolutionary clades. The results revealed that six of the strains are of Colombian origin, while one strain (547256) belongs to the high-risk clone ST773, previously unidentified in Colombia. Genome size ranged from 6 to 7.4 Mbp, indicating differences in genetic content among strains. Phylogenomic analysis confirmed that five strains belong to a multidrug-resistant (MDR) group, while one strain (572897) showed high alignment with a laboratory strain, and strain 547256 was not associated with any specific clade. Clinically, 100% of strains showed carbapenem resistance, resistance genes, and virulence factors that make them difficult to treat. This study provides key insights into the genetic diversity and resistance patterns of P. aeruginosa in this region, underscoring the need to monitor high-risk clones and optimize therapeutic strategies.

Infection or autoinflammatory disease? Difficulties of differential diagnosis on the example of a carrier of the Q705K variant of the NLRP3 gene

The Q705K variant of the NLRP3 gene is considered a variant of uncertain significance and a risk factor contributing to the autoinflammatory disease phenotype. To present a clinical picture of the disease in a pediatric patient carrying the Q705K variant of the NLRP3 gene with a diagnosed cryopyrin-associated periodic syndrome. A 10-year-old child with frequent episodes of fever, refractory to antipyretic therapy, with urticaria, enlarged cervical lymph nodes, manifestations of pharyngitis and frequent respiratory diseases was examined. 338 genes associated with primary immunodeficiency have been analyzed by targeted multigene sequencing. Variant c.2113C>A (p. Gln705Lys) of the NLRP3 gene in a heterozygous state has been identified. The results of laboratory and instrumental research methods during several hospitalizations are analyzed. During hospitalizations with febrile episodes the child was diagnosed, as a rule, with infectious exanthems (yersiniosis, parvovirus, enterovirus, infectious mononucleosis). Chronic persistent herpes type 6 and Epstein-Barr viral infection and a deficiency of cellular immunity have been confirmed. Despite the positive results of the etiological methods, on the example of the last hospitalization - contradictory and not consistent with the clinical picture, given the long history of regular febrile episodes of the same type, accompanied by rashes, a cold reaction, and the identification of an uncertain clinical significance of the NLRP3 gene Q705K in accordance with the accepted diagnostic criteria a child at the age of 18 was diagnosed with cryopyrin-associated periodic syndrome.In a situation of repeated episodes of the same type of febrile disease, the identification of infection markers, especially markers of opportunistic infections, does not exclude autoinflammatory syndrome.

The Pseudomonas aeruginosa T3SS can contribute to traversal of an in situ epithelial multilayer independently of the T3SS needle.

Multilayered epithelia lining our tissue surfaces normally resist traversal by opportunistic bacteria. Previously, we developed a strategy to experimentally perturb this resistance in situ in the corneas of mouse eyes and used it to show that traversal of a multilayered epithelium by Pseudomonas aeruginosa requires ExsA, the transcriptional activator of its type 3 secretion system (T3SS). Here, we developed a novel strategy for quantitatively localizing individual traversing bacteria within the in situ multilayered corneal epithelium and explored the contributions of T3SS components. The results showed that T3SS translocon and T3SS effector mutants had reduced epithelial traversal efficiency. Surprisingly, a ΔpscC mutant unable to assemble the T3SS needle traversed as efficiently as wild-type P. aeruginosa, while a ΔexsD mutant "constitutively on" for T3SS expression was traversal defective. The dispensability of the T3SS needle for effector-mediated traversal was confirmed using a mutant lacking the T3SS operon except for the effector genes (ΔpscU-L mutant). That mutant reacquired the ability to traverse if complemented with rhamnose-inducible exsA, but not if the effector genes were also deleted (ΔpscU-LΔexoSTY). Western immunoblot confirmed ExoS in culture supernatants of rhamnose-induced exsA-complemented ΔpscU-L mutants lacking all T3SS needle protein genes. Together, these results show that epithelial traversal by P. aeruginosa can involve T3SS effectors and translocon proteins independently of the T3SS needle previously thought essential for T3SS function. This advances our understanding of P. aeruginosa pathogenesis and has relevance to the development of therapeutics targeting the T3SS system.IMPORTANCEWhile the capacity to cross an epithelial barrier can be a critical step in bacterial pathogenesis, our understanding of the mechanisms involved is derived largely from cell culture experimentation. The latter is due to the practical limitations of in vivo/in situ models and the challenge of visualizing individual bacteria in the context of host tissue. Here, factors used by P. aeruginosa to traverse an epithelial multilayer in situ were studied by (i) leveraging the transparent properties and superficial location of the cornea, (ii) using our established method for enabling bacterial traversal susceptibility, and (iii) developing a novel strategy for accurate and quantitative localization of individual traversing bacteria in situ. Outcomes showed that T3SS translocon and T3SS effector proteins synergistically contribute to epithelial traversal efficiency independently of the T3SS needle. These findings challenge the assumption that the T3SS needle is essential for T3SS effectors or translocon proteins to contribute to bacterial pathogenesis.

Functional and Pangenomic Exploration of Roc Two-Component Regulatory Systems Identifies Novel Players Across Pseudomonas Species.

The opportunistic pathogen Pseudomonas aeruginosa relies on a large collection of two-component regulatory systems (TCSs) to sense and adapt to changing environments. Among them, the Roc (regulation of cup) system is a one-of-a-kind network of branched TCSs, composed of two histidine kinases (HKs-RocS1 and RocS2) interacting with three response regulators (RRs-RocA1, RocR, and RocA2), which regulate virulence, antibiotic resistance, and biofilm formation. Based on extensive work on the Roc system, previous data suggested the existence of other key regulators yet to be discovered. In this work, we identified PA4080, renamed RocA3, as a fourth RR that is activated by RocS1 and RocS2 and that positively controls the expression of the cupB operon. Comparative genomic analysis of the locus identified a gene-rocR3-adjacent to rocA3 in a subpopulation of strains that encodes a protein with structural and functional similarity to the c-di-GMP phosphodiesterase RocR. Furthermore, we identified a fourth branch of the Roc system consisting of the PA2583 HK, renamed RocS4, and the Hpt protein HptA. Using a bacterial two-hybrid system, we showed that RocS4 interacts with HptA, which in turn interacts with RocA1, RocA2, and RocR3. Finally, we mapped the pangenomic RRs repertoire, establishing a comprehensive view of the plasticity of such regulators among clades of the species. Overall, our work provides a comprehensive inter-species definition of the Roc system, nearly doubling the number of proteins known to be involved in this interconnected network of TCSs controlling pathogenicity in Pseudomonas species.

A Cryptic Incidental Finding of Cryptococcus Lymphadenitis

Abstract Cryptococcus species, particularly C. gattii and C. neoformans, are opportunistic pathogens typically affecting immunocompromised individuals, although cases in immunocompetent patients are increasingly recognized. We report the incidental discovery of cryptococcal lymphadenitis in a hepatic artery lymph node during cancer staging. Differential diagnoses for periportal lymphadenopathy include both malignant and infectious processes, with the presence of necrotizing granulomas narrowing the differential further. This case underscores the importance of considering Cryptococcus in the differential diagnosis of lymphadenopathy, even with a negative cryptococcal antigen and an immunocompetent patient, as early detection and treatment are critical to preventing serious complications.

Valdiazen Derivatives for Chemoproteomic Studies in Burkholderia cenocepacia H111.

Quorum sensing allows bacteria to coordinate community-wide behaviours such as biofilm formation, virulence, and symbiosis. The diazeniumdiolate valdiazen has been identified in the opportunistic pathogen Burkholderia cenocepacia H111 as a novel quorum-sensing signal, yet its protein interactome has remained unexplored. In this study, we employed a chemoproteomic pulldown approach to identify potential valdiazen-binding proteins. For these pulldown experiments, a series of alkyne-linked and biotin-conjugated valdiazen probes were synthesised. Affinity-based pulldown experiments using biotin-valdiazen conjugates successfully identified several putative proteins including an ATP synthase subunit, a succinylglutamate desuccinylase/aspartoacylase, a granule-associated protein, an acetyl-CoA hydrolase, a serine protease and an OmpA/MotB precursor. Overall, this study provides insights into the valdiazen-protein interactome in Burkholderia cenocepacia H111, advancing our understanding of the role of valdiazen in bacterial quorum sensing.

The Prevalence of occurrence of an opportunistic pathogen in Gynmnarchus niloticus, Cuvier (1829) In Anambra River, Nigeria

The available studies have shown that Gynmnarchus niloticus, Cuvier (1829) in Nigerian freshwater bodies record low parasitic infections. Therefore, both parasitic and histopathological studies on the liver of G. niloticus, Cuvier (1829) as one of the most commonly occurring species were carried out to determine the healthy state of these species in the Anambra River after initial observation in one G. niloticus sample from Imo River in 2017. Though no protozoan or helminth infection was detected, there was liver infection by an opportunistic pathogen - Aeromonas sp. 115 samples of G. niloticus were collected and examined for the presence of a pathogen from January to June 2023, 32 (27.8 %) were infected by Aeromonas sp. All the affected G. niloticus were asymptomatic. Sections of the liver showed severe, multifocal, degeneration and necrosis of the hepatocytes with varying degrees of leucocytic/inflammatory cell infiltration. The degenerate hepatocytes showed multiple clear vacuoles in their cytoplasm (micro-vesicular lipidosis). Within the necrotic areas, bacterial colonies consisting of slender short to medium-sized filamentous rods were observed. Clear large vacuoles of varying sizes in the tissue (likely gas bubbles as a result of gas-forming bacterial activity) were also observed. Also, soil nematodes from the soil around the water body were analyzed for heavy metals contents using atomic absorption spectrometry (AAS) mg/kg as a possible source of stress to G. niloticus involving eight elements but three namely Cd (0.005±0.001) mg/kg, Cr (0.0095±0.0015) mg/kg, Pd(0.008±0.000) mg/kg were detected in low, safe levels.

Genome-wide mutagenesis identifies factors involved in MRSA vaginal colonization.

Genome-wide mutagenesis identifies factors involved in MRSA vaginal colonization.

Genetic study of extended spectrum beta-lactamase genes; bla-TEM, blaOXA-10, blaSHV and per-1 in pseudomonas aeruginosa from hospital-acquired infections.

Pseudomonas aeruginosa, an opportunistic pathogen, is a major contributor to hospital-acquired infections. This Gram-negative bacterium relies on aerobic respiration and cannot ferment. Various mechanisms of antibiotic resistance closely link to its pathogenicity. This study aimed to determine how common the bla-TEM, blaOXA-10, blaSHV, and per-1 genes were in Pseudomonas aeruginosa samples taken from hospitalized patients. We conducted a retrospective cross-sectional analysis on 100 isolates of the bacterium. The double-disc test detected extended-spectrum beta-lactamase (ESBL) activity and assessed antibiotic susceptibility. Using polymerase chain reaction (PCR), we found that the bla-TEM, bla-SHV, blaOXA-10, and per-1 genes were present. Pseudomonas aeruginosa isolates were highly resistant to meropenem (80%), imipenem (77%), piperacillin (77%), and ceftazidime (76%). However, they were not resistant to colistin. 91% of the isolates exhibited multidrug resistance (MDR), and 76% phenotypically showed ESBL production.Gene analysis revealed that bla-TEM was the most prevalent (38%), then per-1 (31%), bla-SHV (15%), and OXA-10 (9%). We identified the bla-TEM gene in 50.8% of ESBL-positive Pseudomonas aeruginosa, the per-1 gene in 40.8%, and the bla-SHV and OXA-10 genes in 19.7% and 2.6% respectively. The phenotypic method failed to detect ESBL activity in 24 isolates with the per-1 gene and 7 isolates with the OXA-10 gene. In conclusion, clinical isolates of Pseudomonas aeruginosa show a high prevalence of ESBL activity, but reliance on phenotypic methods alone may overlook some cases. The most frequently detected ESBL-associated genes are bla-TEM, followed by per-1, bla-SHV, and OXA-10. Additionally, Pseudomonas aeruginosa isolates demonstrate a high incidence of multiple drug resistance.

Decoding the genomic landscape of Mammaliicoccus sp. RAM2 isolated from flacherie-infected Bombyx mori L.

Species of the genus Mammaliicoccus are recognized as opportunistic zoonotic pathogens. Here, we report the draft genome sequence of Mammaliicoccus sp. RAM2, isolated from a flacherie-infected Bombyx mori L. (Nistari race) from Raiganj University, India (25.6071° N, 88.1306° E), which offers valuable genetic insights for precise species-level identification.

The development of a bioanalytical method for the simultaneous analysis of gentamicin and tacrolimus in Rat whole blood using UHPLC–MS/MS

Tacrolimus (TAC) is commonly administered to patients who have undergone organ transplantation to prevent the immune system from rejecting the transplanted organ. Multidrug-resistant bacterial infections are the most frequent complications during the first-month post-transplantation. Old antimicrobial agents such as gentamicin (GEN) are widely used to treat opportunistic nosocomial infections in immunosuppressed TAC patients. Nephrotoxicity is a significant side effect of GEN and TAC, but some studies indicated their concurrent administration. However, there is no information on whether the combination of the two drugs may result in a more significant impairment of kidney function than either drug used separately. To investigate this, both drugs should be monitored in blood. Sample preparation was carried out using protein precipitation, requiring only 50 µL of WB sample with an extraction recovery of not less than 95.2% (GEN) and 93.2% (TAC). Analytes and internal standard (IS) were monitored using mass spectrometry (MS) in positive ion mode by multiple reaction monitoring (MRM). Chromatographic analysis was performed on an Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm), kept at 50 °C and using gradient elution. Mobile phase A contained 2 mmol/L ammonium formate acidified with 0.1% formic acid in water, and mobile phase B was a mixture of 2 mmol/L ammonium formate and 0.1% formic acid in methanol, pumped at a flow rate of 0.25 mL/min. The analysis time was only 6 min. The method was verified according to the European Medicines Agency (EMA) guidelines over a concentration range of 19.5–2500 ng/mL for GEN and 1.95–250 ng/mL for TAC. Determination coefficients for the calibration curves were found to be ≥ 0.999. Within- and between-run precision and accuracy were evaluated for both drugs with relative standard deviations (RSD) ≤ 6.5% and inaccuracy ≤ 6.6%. The proposed method was successfully applied to analyze the WB samples at different time points after the co-administration of GEN and TAC to Wistar rats. In this work, a new bioanalytical UHPLC–MS/MS method was developed and validated for simultaneous quantification of total GEN congeners (C1, C1a, and C2/C2a) and TAC in Wistar rats whole blood (WB). The protein precipitation method has been chosen to extract the drug from the WB sample. The assay method has been successfully used to estimate the concentration of TAC and GEN after co-administration in rats.

Hybrid Molecules of Benzothiazole and Hydroxamic Acid as Dual-Acting Biofilm Inhibitors with Antibacterial Synergistic Effect against Pseudomonas aeruginosa Infections.

The ubiquitous opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) causes biofilm-associated drug-resistant infections that often lead to treatment failure. Targeting the bacterium's quorum sensing (QS) and iron homeostasis presents a promising strategy to combat biofilm formation. This study synthesized benzothiazole-conjugated hydroxamic acid derivatives as dual-acting biofilm inhibitors, and compound JH21 was identified as the hit compound with potent submicromolar biofilm inhibitory activity (IC50 = 0.4 μM). Further mechanistic studies demonstrated not only that the production of virulence was decreased through mainly inhibiting QS system but also that JH21 competed for iron with the high-affinity siderophore pyoverdine, inducing iron deficiency and inhibiting biofilm. Moreover, JH21 significantly enhanced the efficacy of tobramycin and ciprofloxacin by 200- and 1000-fold, respectively, in a mouse wound infection model. These results emphasized the feasibility of dual-acting biofilm inhibitors against resistant P. aeruginosa infections and the potential of JH21 as a novel antibacterial synergist.

Flavin-containing siderophore-interacting protein of Shewanella putrefaciens DSM 9451 reveals common structural and functional aspects of ferric-siderophore reduction.

Shewanella are bacteria widespread in marine and brackish water environments and emergent opportunistic pathogens. Their environmental versatility depends on the ability to produce numerous iron-rich proteins, mainly multiheme c-type cytochromes. Although iron plays a vital role in the versatility of Shewanella species, very few studies exist regarding the strategies by which these bacteria scavenge iron from the environment. Siderophore-mediated iron transport is a commonly employed strategy for iron acquisition, and it was identified among Shewanella spp. over two decades ago. Shewanella species produce hydroxamate-type siderophores and iron removal from these compounds can occur in the cytoplasm via Fe(III)-siderophore reduction mediated by siderophore-interacting proteins (SIPs). The genome of Shewanella putrefaciens DSM 9451 isolated from an infected child contains representatives of the two different families of SIPs: the flavin-containing siderophore reductase (SbSIP) and the iron-sulfur cluster-containing ferric-siderophore reductase (SbFSR). Here, we report their expression, purification, and further biochemical characterization of SbSIP. The structural and functional characterization of SbSIP and comparison with the homologous SIP from Shewanella frigidimarina (SfSIP) revealed similarities between these proteins including a common binding pocket for NADH, NADPH, and siderophore substrates plus a pronounced redox-Bohr effect that ensures coupled transfer of electrons and protons in the physiological pH range. These mechanistic aspects open the door for further investigations on developing drugs that interfere with the iron metabolism of these bacteria and thereby prevent their spread.