Characteristics and outcomes of carbapenemase harbouring carbapenem-resistant Klebsiella spp. bloodstream infections: a multicentre prospective cohort study in an OXA-48 endemic setting.

A prospective, multicentre observational cohort study of carbapenem-resistant Klebsiella spp. (CRK) bloodstream infections was conducted in Turkey from June 2018 to June 2019. One hundred eighty-seven patients were recruited. Single OXA-48-like carbapenemases predominated (75%), followed by OXA-48-like/NDM coproducers (16%). OXA-232 constituted 31% of all OXA-48-like carbapenemases and was mainly carried on ST2096. Thirty-day mortality was 44% overall and 51% for ST2096. In the multivariate cox regression analysis, SOFA score and immunosuppression were significant predictors of 30-day mortality and ST2096 had a non-significant effect. All OXA-48-like producers remained susceptible to ceftazidime-avibactam.

Suspension TRAPping Filter (sTRAP) Sample Preparation for Quantitative Proteomics in the Low µg Input Range Using a Plasmid DNA Micro-Spin Column: Analysis of the Hippocampus from the 5xFAD Alzheimer's Disease Mouse Model.

Suspension TRAPping filter (sTRAP) is an attractive sample preparation method for proteomics studies. The sTRAP protocol uses 5% SDS that maximizes protein solubilization. Proteins are trapped on a borosilicate glass membrane filter, where SDS is subsequently removed from the filter. After trypsin digestion, peptides are analyzed directly by LC-MS. Here, we demonstrated the use of a low-cost plasmid DNA micro-spin column for the sTRAP sample preparation of a dilution series of a synapse-enriched sample with a range of 10-0.3 µg. With 120 ng tryptic peptides loaded onto the Evosep LC system coupled to timsTOF Pro 2 mass spectrometer, we identified 5700 protein groups with 4% coefficient of variation (CoV). Comparing other sample preparation protocols, such as the in-gel digestion and the commercial Protifi S-TRAP with the plasmid DNA micro-spin column, the last is superior in both protein and peptide identification numbers and CoV. We applied sTRAP for the analysis of the hippocampal proteome from the 5xFAD mouse model of Alzheimer's disease and their wildtype littermates, and revealed 121 up- and 54 down-regulated proteins. Protein changes in the mutant mice point to the alteration of processes related to the immune system and Amyloid aggregation, which correlates well with the known major Alzheimer's-disease-related pathology. Data are available via ProteomeXchange with the identifier PXD041045.

Higher rates of cefiderocol resistance among NDM producing Klebsiella bloodstream isolates applying EUCAST over CLSI breakpoints.

BACKGROUND: Cefiderocol is generally active against carbapenem-resistant Klebsiella spp. (CRK) with higher MICs against metallo-beta-lactamase producers. There is a variation in cefiderocol interpretive criteria determined by EUCAST and CLSI. Our objective was to test CRK isolates against cefiderocol and compare cefiderocol susceptibilities using EUCAST and CLSI interpretive criteria. METHODS: A unique collection (n = 254) of mainly OXA-48-like- or NDM-producing CRK bloodstream isolates were tested against cefiderocol with disc diffusion (Mast Diagnostics, UK). Beta-lactam resistance genes and multilocus sequence types were identified using bioinformatics analyses on complete bacterial genomes. RESULTS: Median cefiderocol inhibition zone diameter was 24 mm (interquartile range [IQR] 24-26 mm) for all isolates and 18 mm (IQR 15-21 mm) for NDM producers. We observed significant variability between cefiderocol susceptibilities using EUCAST and CLSI breakpoints, such that 26% and 2% of all isolates, and 81% and 12% of the NDM producers were resistant to cefiderocol using EUCAST and CLSI interpretive criteria, respectively. CONCLUSIONS: Cefiderocol resistance rates among NDM producers are high using EUCAST criteria. Breakpoint variability may have significant implications on patient outcomes. Until more clinical outcome data are available, we suggest using EUCAST interpretive criteria for cefiderocol susceptibility testing.

Efficacy of Amikacin and Meropenem on Colistin-Induced Klebsiella pneumoniae Persisters.

Colistin-based antibiotic therapies have been recommended for the treatment of multidrug-resistant Klebsiella pneumoniae infections. During colistin treatment, persister cells that tolerate antibiotics may arise. Here we designed an in vitro study to assess the killing activity of colistin, meropenem, and amikacin on colistin-induced K. pneumoniae persisters in comparison with starvation-induced persisters. Colistin-induced persisters were generated under exposure to 10 × minimum inhibitory concentration dose of colistin, whereas starvation-induced persisters were produced by limitation of nutrients. In colistin-induced persisters, amikacin totally inhibited cell growth in 6 hours, whereas 98% of the cell population was inhibited by meropenem, and total eradication with meropenem was observed after 24 hours. Both antibiotics also inhibited metabolic activity >88%. The lack of killing effect under colistin exposure suggested to us that these cells could protect themselves from further colistin stress. There was no significant permeabilization change in the cellular membrane with all antibiotics. There was no killing effect on starvation-induced persister cells with the exposure to all antibiotics. In 6 hours, the metabolic activity of the persisters with meropenem and colistin increased 99% and 40%, respectively, whereas there was no increase with amikacin. The sustained inhibition with amikacin was an important finding for antipersister effect of amikacin. Amikacin had rapid and sustained antipersister activity on colistin-induced persister cells. During the colistin treatment of K. pneumoniae infection, the addition of amikacin to the regimen seems to be an effective approach to prevent a recurrence.

Protein Scaffold-Based Multimerization of Soluble ACE2 Efficiently Blocks SARS-CoV-2 Infection In Vitro and In Vivo.

Soluble ACE2 (sACE2) decoys are promising agents to inhibit SARS-CoV-2, as their efficiency is unlikely to be affected by escape mutations. However, their success is limited by their relatively poor potency. To address this challenge, multimeric sACE2 consisting of SunTag or MoonTag systems is developed. These systems are extremely effective in neutralizing SARS-CoV-2 in pseudoviral systems and in clinical isolates, perform better than the dimeric or trimeric sACE2, and exhibit greater than 100-fold neutralization efficiency, compared to monomeric sACE2. SunTag or MoonTag fused to a more potent sACE2 (v1) achieves a sub-nanomolar IC50 , comparable with clinical monoclonal antibodies. Pseudoviruses bearing mutations for variants of concern, including delta and omicron, are also neutralized efficiently with multimeric sACE2. Finally, therapeutic treatment of sACE2(v1)-MoonTag provides protection against SARS-CoV-2 infection in an in vivo mouse model. Therefore, highly potent multimeric sACE2 may offer a promising treatment approach against SARS-CoV-2 infections.

Comparison of ceftazidime-avibactam susceptibility testing methods against OXA-48-like carrying Klebsiella blood stream isolates.

Ceftazidime-avibactam exhibits good in vitro activity against carbapenem resistant Klebsiella carrying OXA-48-like enzymes. We tested two hundred unique carbapenem resistant Klebsiella blood stream isolates (71% with single OXA-48-like carbapenemases, including OXA-48, n = 62; OXA-232, n = 57; OXA-244, n = 17; OXA-181, n = 5) that were collected as part of a multicentre study against ceftazidime-avibactam using Etest (bioMérieux, Marcyl'Étoile, France), 10/4 µg disc (Thermo Fisher) and Sensititre Gram Negative EURGNCOL Plates (Lyophilized panels, Sensititre, Thermo Fisher) with the aim of comparing the performances of the Etest and disc to that of Sensititre. Ceftazidime-avibactam MIC50/90 was 2/>16 mg/L for the entire collection and was 2/4 mg/L for single OXA-48-like producers. Categorical and essential agreements between the Etest and Sensititre were 100% and 97%, respectively. Categorical agreement between the disc and Sensititre was 100%. Etest and 10/4 µg discs are suitable alternatives to Sensititre for ceftazidime-avibactam sensitivity testing for OXA-48-like producers.

High prevalence of ArmA-16S rRNA methyltransferase among aminoglycoside-resistant Klebsiella pneumoniae bloodstream isolates.

Introduction. Aminoglycosides are used for the treatment of carbapenemase-producing Klebsiella pneumoniae (CPK) infections. 16S rRNA methyltransferases (RMTs) confer resistance to all aminoglycosides and are often cocarried with NDM.Hypothesis/Gap Statement. There is a dart of studies looking at the aminoglycoside resistance mechanisms for invasive CPK isolates, particularly in OXA-48 endemic settings.Aim. We aimed to determine the prevalence of RMTs and their association with beta lactamases and MLSTs amongst aminoglycoside-resistant CPK bloodstream isolates in an OXA-48 endemic setting.Methodology. CPK isolates (n=181), collected as part of a multicentre cohort study, were tested for amikacin, gentamicin and tobramycin susceptibility using custom-made sensititre plates (GN2XF, Thermo Fisher Scientific). All isolates were previously subjected to whole-genome sequencing. Carbapenemases, RMTs, MLSTs and plasmid incompatibility groups were detected on the assembled genomes.Results. Of the 181 isolates, 109(60 %) were resistant to all three aminoglycosides, and 96 of 109(88 %) aminoglycoside-resistant isolates carried an RMT (85 ArmA, 10 RmtC, 4 RmtF1; three isolates cocarried ArmA and RmtC). Main clonal types associated with ArmA were ST2096 (49/85, 58 %) and ST14 (24/85, 28 %), harbouring mainly OXA-232 and OXA-48 +NDM, respectively. RmtC was cocarried with NDM (5/10) on ST395, and NDM +OXA-48 or NDM +KPC (4/10) on ST14, ST15 and ST16. All RMT producers also carried CTX-M-15, and the majority cocarried SHV-106, TEM-150 and multiple other antibiotic resistance genes. The majority of the isolates harboured a combination of IncFIB, IncH and IncL/M type plasmids. Non-NDM producing isolates remained susceptible to ceftazidime-avibactam.Conclusion. Aminoglycoside resistance amongst CPK bloodstream isolates is extremely common and mainly driven by clonal spread of ArmA carried on ST2096 and ST14, associated with OXA-232 and OXA48 +NDM carriage, respectively.

Effects of Type 2 Diabetes Mellitus on Gene Expressions of Mouse Meibomian Glands.

Purpose: Type 2 Diabetes mellitus (DM) is a major health problem and its ocular complications like orbital infections, cataract and diabetic retinopathy cause blindness. Meibomian gland (MG) dysfunction and dry eye disease are also important ocular complications of type 2 DM but not enough research has been conducted on these complications. Our hypothesis suggests type 2 DM can alter significant gene expressions of MG. In our study, MGs of leptin-deficient spontaneous diabetic and non-diabetic mice were extracted, and gene expression profiles were analyzed with microarray technology.Methods: Mice were divided into two groups; nine Lep b/ob spontaneous diabetic mice as type 2 DM group and nine non-diabetic Balb/c mice as controls. Blood glucose levels, tearfilm break-up time and fluorescein scores were measured in both two groups for 12 weeks. MGs were dissected and RNAs were isolated for microarray gene expression analysis. We filtered probes with standard deviation of more than 0.1 and we used 40452 of 45281 probes for processing. We performed fold change analysis and identified which genes are affected, and we analyzed the impact of genes on proteins, pathways and gene ontologies by using various databases.Results: We observed 172 up-regulated and 118 down-regulated genes in type 2 diabetic mice when compared to non-diabetic mice. Interestingly, expression of collagen type I, integrin beta-I binding protein-I, pyruvate dehydrogenase kinase, TNF receptor genes up-regulated with DM; on the other hand, IL-33, cholecystokinin, plasminogen activator, IL-1 and serine peptidase inhibitor genes down-regulated significantly. Also, we have seen a significant decrease in WNT signaling and pentose phosphate pathways-related genes.Conclusion: Our data show these changes in gene expression caused by endocrine and immune mechanisms of type 2 DM which result disrupted homeostasis of epithelial cells of MG. Increased expressions of apoptosis and inflammation-related genes and their effects on related pathways have proven that MGs were negatively affected by type-2 DM.

Androgen Suppresses Hyperosmolarity-Induced Inflammatory Mediators in Human Corneal Epithelial Cells.

PURPOSE: To investigative the effects of sex steroids on hyperosmolar stress-induced proinflammatory cytokine expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-8, and IL-6, and on the mitogen-activated protein kinase pathway in immortalized human corneal epithelial cells (hCECs). METHODS: Immortalized hCECs were cultured with keratinocyte-free medium until reaching 80% confluency with either 10 M dihidrotestosteron (DHT) or 10 M 17-ß-estradiol, and then, the medium was changed to hyperosmolar for various time points. After hyperosmolar treatment, a real-time polymerase chain reaction was performed to show the TNF-α, IL-8, and IL-6 gene expression levels in hCECs. In addition, the treated cells were lysed, and Western blot analysis was applied for phosphorylated and nonphosphorylated forms of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 kinase, and c-Jun N-terminal kinase 1/2 (JNK1/2). hCECs viability was measured with Annexin V/propidium iodide. RESULTS: Pretreatment with 10 M DHT or 17-ß-estradiol inhibited the high osmolarity-induced expression of TNF-α, IL-8, and IL-6. The upregulation of p-ERK, p-JNK, and p-p38 with high osmolarity was inhibited partially by DHT, but 17-ß-estradiol pretreatment only affected p-p38 for a short time interval. In addition, DHT increased cell viability of hCECs under hyperosmolar conditions. CONCLUSIONS: Our results demonstrated that DHT and 17-ß-estradiol decreased the proinflammatory cytokine gene expression levels which were stimulated by high osmolarity in immortalized hCECs. The mitogen-activated protein kinase signaling pathway is partially involved in the regulatory effects of DHT on hCECs. These findings may contribute to the etiologic role and therapeutic implications of sex steroids in certain ocular surface diseases.

Co-existence of OXA-48 and NDM-1 in colistin resistant Pseudomonas aeruginosa ST235.

Here, we presented 11 cases with colistin-resistant Pseudomonas aeruginosa infection and co-existence of OXA-48 and NDM-1 in the ST235 high-risk clone. The molecular analyses were performed by Sanger sequencing and RT-PCR. The eight patients (72.7%) had an invasive infection and three (27.3%) had colonization. The 30-day mortality rate was 87.5% (7/8). Three patients (37.5%, 3/8) received colistin therapy before isolation of P. aeruginosa. In the Multilocus sequence typing (MLST) analysis of 11 isolates, eight (72.7%) isolates belonged to P. aeruginosa ST235 clone. All isolates were NDM-1 positive, and nine isolates (81.8%) were found to be positive for both OXA-48 and NDM-1. Sequences of pmrAB and phoPQ revealed numerous insertions and deletions in all isolates. In 10 isolates pmrAB and phoPQ were found to be upregulated. In conclusion, the co-existence of OXA-48 and NDM-1 genes in colistin-resistant P. aeruginosa ST235 high-risk clone indicates the spread of carbapenemases in clinical isolates and highlights need of continuous surveillance for high-risk clones of P. aeruginosa.