Important Determinant Of Resistance Research Articles

More than just the gene: investigating expression using a non-native plasmid and host and its impact on resistance conferred by β-lactamase OXA-58 isolated from a hospital wastewater microbiome.

With the escalation of hospital-acquired infections by multidrug resistant bacteria, understanding antibiotic resistance is of paramount importance. This study focuses on the β-lactamase gene, blaOXA-58, an important resistance determinant identified in a patient-facing hospital wastewater system. This study aimed to characterize the behaviour of the OXA-58 enzyme when expressed using a non-native plasmid and expression host. blaOXA-58 was cloned using a pET28a(+)/Escherichia coli BL21(DE3) expression system. Nitrocefin hydrolysis and antimicrobial susceptibility of OXA-58-producing cells were assessed against penicillin G, ampicillin, meropenem, and amoxicillin. blaOXA-58 conferred resistance to amoxicillin, penicillin G, and ampicillin, but not to meropenem. This was unexpected given OXA-58's annotation as a carbapenemase. The presence of meropenem also reduced nitrocefin hydrolysis, suggesting it acts as a competitive inhibitor of the OXA-58 enzyme. This study elucidates the phenotypic resistance conferred by an antimicrobial resistance gene (ARG) obtained from a clinically relevant setting and reveals that successful functional expression of ARGs is multifaceted. This study challenges the reliability of predicting antimicrobial resistance based solely on gene sequence alone, and serves as a reminder of the intricate interplay between genetics and structural factors in understanding resistance profiles across different host environments.

A new type of Class C β-lactamases defined by PIB-1. A metal-dependent carbapenem-hydrolyzing β-lactamase, from Pseudomonas aeruginosa: Structural and functional analysis

Antibiotic resistance is one of most important health concerns nowadays, and β-lactamases are the most important resistance determinants. These enzymes, based on their structural and functional characteristics, are grouped in four categories (A, B, C and D). We have solved the structure of PIB-1, a Pseudomonas aeruginosa chromosomally-encoded β-lactamase, in its apo form and in complex with meropenem and zinc. These crystal structures show that it belongs to the Class C β-lactamase group, although it shows notable differences, especially in the Ω- and P2-loops, which are important for the enzymatic activity. Functional analysis showed that PIB-1 is able to degrade carbapenems but not cephalosporins, the typical substrate of Class C β-lactamases, and that its catalytic activity increases in the presence of metal ions, especially zinc. They do not bind to the active-site but they induce the formation of trimers that show an increased capacity for the degradation of the antibiotics, suggesting that this oligomer is more active than the other oligomeric species. While PIB-1 is structurally a Class C β-lactamase, the low sequence conservation, substrate profile and its metal-dependence, prompts us to position this enzyme as the founder of a new group inside the Class C β-lactamases. Consequently, its diversity might be wider than expected.

Overexpression of a plasmalemma Na+/H+ antiporter from the halophyte Nitraria sibirica enhances the salt tolerance of transgenic poplar

The plasmalemma Na+/H+ antiporter Salt Overly Sensitive 1 (SOS1) is responsible for the efflux of Na+ from the cytoplasm, an important determinant of salt resistance in plants. In this study, an ortholog of SOS1, referred to as NsSOS1, was cloned from Nitraria sibirica, a typical halophyte that grows in deserts and saline-alkaline land, and its expression and function in regulating the salt tolerance of forest trees were evaluated. The expression level of NsSOS1 was higher in leaves than in roots and stems of N. sibirica, and its expression was upregulated under salt stress. Histochemical staining showed that β-glucuronidase (GUS) driven by the NsSOS1 promoter was strongly induced by abiotic stresses and phytohormones including salt, drought, low temperature, gibberellin, and methyl jasmonate, suggesting that NsSOS1 is involved in the regulation of multiple signaling pathways. Transgenic 84 K poplar (Populus alba × P. glandulosa) overexpressing NsSOS1 showed improvements in survival rate, root biomass, plant height, relative water levels, chlorophyll and proline levels, and antioxidant enzyme activities versus non-transgenic poplar (NT) under salt stress. Transgenic poplars accumulated less Na+ and more K+ in roots, stems, and leaves, which had a lower Na+/K+ ratio compared to NT under salt stress. These results indicate that NsSOS1-mediated Na+ efflux confers salt tolerance to transgenic poplars, which show more efficient photosynthesis, better scavenging of reactive oxygen species, and improved osmotic adjustment under salt stress. Transcriptome analysis of transgenic poplars confirmed that NsSOS1 not only mediates Na+ efflux but is also involved in the regulation of multiple metabolic pathways. The results provide insight into the regulatory mechanisms of NsSOS1 and suggest that it could be used to improve the salt tolerance of forest trees.

Genetic diversity, gene flow, and landscape resistance in a pond-breeding amphibian in agricultural and natural forested landscapes in Norway.

Genetic diversity is a key part of biodiversity, threatened by human activities that lead to loss of gene flow and reduction of effective population sizes. Gene flow is a result of both landscape connectivity and demographic processes determining the number of dispersing individuals in space and time. Thus, the effect of human impact on processes determining the level of genetic diversity must be interpreted in the context of basic ecological conditions affecting survival and recruitment. When the intensity of human impact and habitat suitability correlate, the effect on genetic diversity and gene flow may be challenging to predict. We compared genetic diversity, gene flow and landscape resistance in two contrasting landscapes in Norway for the pond-breeding amphibian Triturus cristatus: a highly human-impacted, agricultural landscape with ecologically productive habitats, and a forested landscape with less productive habitats and lower levels of human impact. Our results show that genetic diversity was higher and gene flow lower within the forested landscape. Microclimatic moisture conditions and vegetation cover were important determinants of landscape resistance to gene flow within both landscapes. There were indications that landscape resistance was increased by minor roads in the forested landscape, which was not the case for the agricultural landscape, suggesting a higher vulnerability to human interference within the landscape matrix for the populations in less productive habitats. Our findings suggest that the effect of human impact on genetic diversity may not be straightforward but modulated by the ecological conditions underlying local demographic processes. Populations within both landscapes seem to be vulnerable to loss of genetic diversity, but due to different mechanisms. This has implications for the choice of relevant management actions, that is, increasing population stability may be more relevant within an agricultural landscape still permeable for dispersal, while conserving dispersal corridors may be more appropriate in the forested landscape, to avoid isolation and increased genetic drift.

Pangenomic analyses of antibiotic-resistant Campylobacter jejuni reveal unique lineage distributions and epidemiological associations.

Application of whole-genome sequencing (WGS) to characterize foodborne pathogens has advanced our understanding of circulating genotypes and evolutionary relationships. Herein, we used WGS to investigate the genomic epidemiology of Campylobacter jejuni, a leading cause of foodborne disease. Among the 214 strains recovered from patients with gastroenteritis in Michigan, USA, 85 multilocus sequence types (STs) were represented and 135 (63.1 %) were phenotypically resistant to at least one antibiotic. Horizontally acquired antibiotic resistance genes were detected in 128 (59.8 %) strains and the genotypic resistance profiles were mostly consistent with the phenotypes. Core-gene phylogenetic reconstruction identified three sequence clusters that varied in frequency, while a neighbour-net tree detected significant recombination among the genotypes (pairwise homoplasy index P<0.01). Epidemiological analyses revealed that travel was a significant contributor to pangenomic and ST diversity of C. jejuni, while some lineages were unique to rural counties and more commonly possessed clinically important resistance determinants. Variation was also observed in the frequency of lineages over the 4 year period with chicken and cattle specialists predominating. Altogether, these findings highlight the importance of geographically specific factors, recombination and horizontal gene transfer in shaping the population structure of C. jejuni. They also illustrate the usefulness of WGS data for predicting antibiotic susceptibilities and surveillance, which are important for guiding treatment and prevention strategies.

Association between cardiorespiratory fitness level and insulin resistance in adolescents with various obesity categories.

An association between cardiorespiratory fitness (CRF) and insulin resistance in obese adolescents, especially in those with various obesity categories, has not been systematically studied. There is a lack of knowledge about the effects of CRF on insulin resistance in severely obese adolescents, despite their continuous rise. To investigate the association between CRF and insulin resistance in obese adolescents, with special emphasis on severely obese adolescents. We performed a prospective, cross-sectional study that included 200 pubertal adolescents, 10 years to 18 years of age, who were referred to a tertiary care center due to obesity. According to body mass index (BMI), adolescents were classified as mildly obese (BMI 100% to 120% of the 95th percentile for age and sex) or severely obese (BMI ≥ 120% of the 95th percentile for age and sex or ≥ 35 kg/m2, whichever was lower). Participant body composition was assessed by bioelectrical impedance analysis. A homeostatic model assessment of insulin resistance (HOMA-IR) was calculated. Maximal oxygen uptake (VO2max) was determined from submaximal treadmill exercise test. CRF was expressed as VO2max scaled by total body weight (TBW) (mL/min/kg TBW) or by fat free mass (FFM) (mL/min/kg FFM), and then categorized as poor, intermediate, or good, according to VO2max terciles. Data were analyzed by statistical software package SPSS (IBM SPSS Statistics for Windows, Version 24.0). P < 0.05 was considered statistically significant. A weak negative correlation between CRF and HOMA-IR was found [Spearman's rank correlation coefficient (rs) = -0.28, P < 0.01 for CRFTBW; (rs) = -0.21, P < 0.01 for CRFFFM]. One-way analysis of variance (ANOVA) revealed a significant main effect of CRF on HOMA-IR [F(2200) = 6.840, P = 0.001 for CRFTBW; F(2200) = 3.883, P = 0.022 for CRFFFM]. Subsequent analyses showed that obese adolescents with poor CRF had higher HOMA-IR than obese adolescents with good CRF (P = 0.001 for CRFTBW; P = 0.018 for CRFFFM). Two-way ANOVA with Bonferroni correction confirmed significant effect of interaction of CRF level and obesity category on HOMA-IR [F(2200) = 3.292, P = 0.039 for CRFTBW]. Severely obese adolescents had higher HOMA-IR than those who were mildly obese, with either good or poor CRF. However, HOMA-IR did not differ between severely obese adolescents with good and mildly obese adolescents with poor CRF. CRF is an important determinant of insulin resistance in obese adolescents, regardless of obesity category. Therefore, CRF assessment should be a part of diagnostic procedure, and its improvement should be a therapeutic goal.

Expansion of healthcare-associated hypervirulent KPC-2-producing Klebsiella pneumoniae ST11/KL64 beyond hospital settings

The spread of carbapenemase-producing Klebsiella pneumoniae beyond hospital settings is a global critical issue within a public health and One Health perspective. Another worrisome concern is the convergence of virulence and resistance in healthcare-associated lineages of K. pneumoniae leading to unfavorable clinical outcomes. During a surveillance study of WHO critical priority pathogens circulating in an impacted urban river in São Paulo, Brazil, we isolate two hypermucoviscous and multidrug-resistant K. pneumoniae strains (PINH-4250 and PINH-4900) from two different locations near to medical centers. Genomic investigation revealed that both strains belonged to the global high-risk sequence type (ST) ST11, carrying the blaKPC-2 carbapenemase gene, besides other medically important antimicrobial resistance determinants. A broad virulome was predicted and associated with hypervirulent behavior in the Galleria mellonella infection model. Comparative phylogenomic analysis of PINH-4250 and PINH-4900 along to an international collection of publicly available genomes of K. pneumoniae ST11 revealed that both environmental strains were closely related to hospital-associated K. pneumoniae strains recovered from clinical samples between 2006 and 2018, in São Paulo city. Our findings support that healthcare-associated KPC-2-positive K. pneumoniae of ST11 clone has successfully expanded beyond hospital settings. In summary, aquatic environments can become potential sources of international clones of K. pneumoniae displaying carbapenem resistance and hypervirulent behaviors, which is a critical issue within a One Health perspective.

1630-P: Reduced Fatty Acid Oxidation Does Not Prevent Intralipid-Induced Peripheral Insulin Resistance in Humans

Mitochondrial handling of excess lipid substrate is thought to be an important determinant of insulin resistance. Evidence from animal models support insulin signaling defects resulting from either intra-cellular lipid accumulation or by mitochondrial dysfunction and altered fatty acid oxidation (FAO) flux. To explore the relationship between FAO and insulin sensitivity, we measured the change in peripheral glucose disposal (Rd) during a 5-hour hyperinsulinemic euglycemic clamp with co-infusion of a control glycerol solution versus a 20% intralipid solution in 18 subjects with an inherited defect in FAO (FAOD; including Carnitine Palmitoyltransferase 2, Very long-chain, medium-chain or long-chain 3-hydroxy AcylcoA Dehydrogenase Deficiencies) and 18 age, sex, and weight-matched normal healthy controls. Rd was similar between groups during the glycerol control clamp. Intramyocellular lipid (IMCL) content as measured by MRS increased after the 5 hour intralipid infusion in controls but not in participants with FAOD. Plasma free fatty acids during intralipid infusion were higher in FAOD compared to controls suggesting infused lipid remained in circulation. Muscle fiber type and expression of lipid transporters CD36 and FATP1 were similar in biopsy samples between groups and cannot explain decreased lipid uptake into muscle among FAOD subjects. However, intralipid suppressed Rd equally in subjects with FAOD and controls inducing peripheral insulin resistance in both groups. Impaired FAO decreased lipid accumulation in the muscle during intralipid infusion, but despite lower IMCL, subjects with FAOD developed peripheral insulin resistance similar to controls. Increased IMCL is not essential for, nor is reduced FAO flux protective against, the development of peripheral insulin resistance during intralipid infusion. Disclosure M.B.Gillingham: None. J.Q.Purnell: Advisory Panel; Boehringer Ingelheim International GmbH, Novo Nordisk. A.N.Gregor: None. O.Varlamov: None. J.J.Robino: None. C.E.Mccurdy: None. K.T.Greyslak: None. D.Choi: None. E.Baetscher: Consultant; Biogen. W.Rooney: Consultant; Ultragenix, Research Support; Revalesio. Funding National Institutes of Health (DK102813)

Role of obesity related inflammation in pathogenesis of peripheral artery disease in patients of type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) has emerged as one of the greatest global health challenges of twenty-first century. Visceral obesity is one of the most important determinant of insulin resistance (IR) as well as T2DM complications. Therefore this review focuses on the molecular mechanism of obesity induced inflammation, signaling pathways contributing to diabetes, as well as role of lifestyle interventionsand medical therapies in the prevention and management of T2DM. Articles were searched on digital data base PubMed, Cochrane Library, and Web of Science. The key words used for search included Type 2 diabetes mellitus, obesity, insulin resistance, vascular inflammation and peripheral arterial disease. Visceral obesity is associated with chronic low grade inflammation and activation of immune systems which are involved in pathogenesis of obesity related IR and T2DM. Metabolic dysregulation of adipose tissue leads to local hypoxia, misfolded/unfolded protein response and increased circulating free fatty acids, which in turn initiate inflammatory signaling cascades in the population of infiltrating cells. Mechanism that relates the role of adipocytokines with insulin sensitivity and glucose homeostasis might throw a light on the development of therapeutic interventions and subsequently might result in the reduction of vascular complications.

Maternal Preconception Body Mass Index Overtakes Age as a Risk Factor for Gestational Diabetes Mellitus.

Introduction-The purpose of this study was to determine the relative impact of modifiable and non-modifiable risk factors in the development of gestational diabetes mellitus (GDM), with a particular focus on maternal preconception body mass index (BMI) and age, two important determinants of insulin resistance. Understanding the factors that contribute most to the current escalation of GDM rates in pregnant women could help to inform prevention and intervention strategies, particularly in areas where this female endocrine disorder has an elevated prevalence. Methods-A retrospective, contemporary, large population of singleton pregnant women from southern Italy who underwent 75 g OGTT for GDM screening was enrolled at the Endocrinology Unit, "Pugliese Ciaccio" Hospital, Catanzaro. Relevant clinical data were collected, and the characteristics of women diagnosed with GDM or with normal glucose tolerance were compared. The effect estimates of maternal preconception BMI and age as risk factors for GDM development were calculated through correlation and logistic regression analysis by adjusting for potential confounders. Results-Out of the 3856 women enrolled, 885 (23.0%) were diagnosed with GDM as per IADPSG criteria. Advanced maternal age (≥35 years), gravidity, reproductive history of spontaneous abortion(s), previous GDM, and thyroid and thrombophilic diseases, all emerged as non-modifiable risk factors of GDM, whereas preconception overweight or obesity was the sole potentially modifiable risk factor among those investigated. Maternal preconception BMI, but not age, had a moderate positive association with fasting glucose levels at the time of 75 g OGTT (Pearson coefficient: 0.245, p < 0.001). Abnormalities in fasting glucose drove the majority (60%) of the GDM diagnoses in this study. Maternal preconception obesity almost tripled the risk of developing GDM, but even being overweight resulted in a more pronounced increased risk of developing GDM than advanced maternal age (adjusted OR for preconception overweight: 1.63, 95% CI 1.320-2.019; adjusted OR for advanced maternal age: 1.45, 95% CI 1.184-1.776). Conclusions-Excess body weight prior to conception leads to more detrimental metabolic effects than advanced maternal age in pregnant women with GDM. Thus, in areas in which GDM is particularly common, such as southern Italy, measures aiming to counteracting maternal preconception overweight and obesity may be efficient in reducing GDM prevalence.

Identification of Prognostic and Predictive Biomarkers and Druggable Targets among 205 Antioxidant Genes in 21 Different Tumor Types via Data-Mining.

(1) Background: Oxidative stress is crucial in carcinogenesis and the response of tumors to treatment. Antioxidant genes are important determinants of resistance to chemotherapy and radiotherapy. We hypothesized that genes involved in the oxidative stress response may be valuable as prognostic biomarkers for the survival of cancer patients and as druggable targets. (2) Methods: We mined the KM Plotter and TCGA Timer2.0 Cistrome databases and investigated 205 antioxidant genes in 21 different tumor types within the context of this investigation. (3) Results: Of 4347 calculations with Kaplan-Meier statistics, 84 revealed statistically significant correlations between high gene expression and worse overall survival (p < 0.05; false discovery rate ≤ 5%). The tumor types for which antioxidant gene expression was most frequently correlated with worse overall survival were renal clear cell carcinoma, renal papillary cell carcinoma, and hepatocellular carcinoma. Seventeen genes were clearly overexpressed in tumors compared to their corresponding normal tissues (p < 0.001), possibly qualifying them as druggable targets (i.e., ALOX5, ALOX5AP, EPHX4, G6PD, GLRX3, GSS, PDIA4, PDIA6, PRDX1, SELENOH, SELENON, STIP1, TXNDC9, TXNDC12, TXNL1, TXNL4A, and TXNRD1). (4) Conclusions: We concluded that a sub-set of antioxidant genes might serve as prognostic biomarkers for overall survival and as druggable targets. Renal and liver tumors may be the most suitable entities for this approach.

Mutations in atpG2 may confer resistance to gentamicin in Listeria monocytogenes.

Introduction Listeriosis, a foodborne infection caused by Listeria monocytogenes, could lead to febrile listerial gastroenteritis and a more invasive form which is often associated with a high mortality and hospitalisation rate. Gentamicin, used as an adjunct therapy with ampicillin, remains the treatment of choice for this life-threatening and invasive infection.Gap statement Nevertheless, there is little data on gentamicin resistance determinants in L. monocytogenes.Aim In this study, we selected and characterised B2b, a gentamicin-resistant mutant derived from L. monocytogenes ATCC 19115 to determine the target(s) of resistance in L. monocytogenes after exposure to gentamicin.Methodology Whole-genome sequencing was carried out to identify the mutation site(s) and possible mechanism(s) of resistance. The mutant was characterised using antimicrobial susceptibility testing and PCR. For biological verifications, complementation and allelic exchange mutagenesis were carried out.Results We found that the gentamicin resistance in B2b was caused by a 10 bp deletion in atpG2 which encodes a gamma subunit of the ATP synthase in L. monocytogenes. Using atpG2 PCR, various other mutations were identified in other gentamicin resistant mutants derived from ATCC 19115. In addition, the mutation from B2b, when introduced into L. ivanovii, also caused gentamicin resistance in this Listeria species.Conclusion Hence, atpG2 mutations appear to be important determinants of gentamicin resistance not only in L. monocytogenes but possibly also in other Listeria species.

Stress resistance as a feature of character of the Ukrainians

The article discloses the peculiarities of stress resistance of the Ukrainian ethnicity. Has been analyzed the importance of the national character of Ukrainians as an important determinant of stress resistance. Also, the main features of the Ukrainian national character, which are related to stress resistance have been highlighted. The significance of the collective traumatic experience as a factor in increasing vulnerability to stress has been considered. The author includes sensitivity, cordocentrism, individualism, freedom-loving, intolerance to coercion and violence, hard work, antaeuism, and optimism to the features of the national character that are associated with stress resistance. Emphasis is placed on the positive or ambiguous relationship of selected features with stress resistance. A positive or ambiguous relationship of the selected features with stress resistance was noted. The propensity of Ukrainians to provide social support in crisis situations has been disclosed, which has a significant positive effect on the psychological well-being of large groups. In the opinion of the author, taking into account the previous and past historical experience of the Ukrainian ethnicity, collective trauma appears to be a growing problem, which has a long-term negative impact on psychological well-being and stress resistance

The Effects of Phyllosphere Bacteria on Plant Physiology and Growth of Soybean Infected with Pseudomonas syringae.

Phyllosphere bacteria are an important determinant of plant growth and resistance to pathogens. However, the efficacy of phyllosphere bacteria in regulating infection of Pseudomonas syringae pv. glycinea (Psg) and its influence on soybean growth and physiology is unknown. In a greenhouse study, we assessed the influence of a phyllosphere bacterial consortium (BC) of 13 species isolated from field-grown soybean leaves on uninfected and deliberately Psg infected soybean plants. We measured Psg density on infected leaves with and without the application of the BC. The BC application resulted in a significant reduction in Psg cells. We also measured plant biomass, nodule mass and number, gas exchange, and leaf chlorophyll and nitrogen in four treatment groups: control plants, plants with a BC and no infection (BC), plants with BC and infected with Psg (BC + Psg), and plants infected with Psg alone. For all variables, plants infected with Psg alone showed significant reduction in measured variables compared to both BC treatments. Therefore, the bacterial consortium was effective in controlling the negative effects of Psg on growth and physiology. The BC treatment sometimes resulted in increases in measured variables such as plant biomass, nodule numbers, and leaf chlorophyll as compared to control and BC + Psg treatments. Overall, the positive influence of BC treatment on plant growth and physiology highlights its potential applications to increase crop yield and control bacterial pathogens.

Distribution and spread of the mobilised RND efflux pump gene cluster tmexCD-toprJ in clinical Gram-negative bacteria: a molecular epidemiological study

TMexCD1-TOprJ1, which is associated with phenotypic resistance to multiple classes of antibiotics, is a transmissible resistance-nodulation-division (RND) family efflux pump. However, the prevalence and genomic and phenotypic characteristics of clinical isolates with this important resistance determinant are poorly understood. In this study, we aimed to survey tmexCD-toprJ among clinical Gram-negative isolates collected from hospitals in China between 1991 and 2020 and characterise tmexCD-toprJ-positive clinical isolates. We conducted online data retrieval and active nationwide surveillance in China to screen tmexCD-toprJ-positive strains. We characterised tmexCD-toprJ-positive clinical strains for their antimicrobial susceptibility, genetic and functional characteristics, and the potential inter-species transmission route of tmexCD-toprJ with whole genome sequencing and bioinformatics analyses. The function of tmexCD-toprJ in Pseudomonas sp and Proteus sp was investigated by tmexD gene knockdown using an isopropylthio-β-galactoside-inducible CRISPR interference system. Data retrieval obtained 53 strains carrying tmexCD-toprJ, comprising 32 Pseudomonas spp, 11 Klebsiella pneumoniae, one Aeromonas spp, one Citrobacter freundii, and one uncultured bacterium from diverse niches. 48 (0·64%) of 7517 clinical isolates from China, including seven Klebsiella spp, one Proteus mirabilis, and 40 Pseudomonas spp, carried tmexCD-toprJ. These isolates exhibited multidrug resistance phenotypes and co-harboured resistance genes, such as mcr and carbapenemases genes. tmexCD-toprJ was encoded on both plasmids and chromosomes in all Klebsiella spp that carried plasmid-borne tmexCD-toprJ (n=7), P mirabilis carried chromosome-borne tmexCD-toprJ, and Pseudomonas spp carried either plasmid-borne (n=19) or chromosome-borne (n=21) ones. tmexCD-toprJ had undergone clonal and horizontal transmission among clinical pathogens. Eight different types of genetic context of tmexCD-toprJ were identified, each of which was associated with different mobile elements, including IntI, IS6100, TnAs1-like, ISRor5, ISVsa3, ISCfr-like, Tn5393, and IS222-like, which might facilitate its transmission. Knockdown of tmexD led to a four times decrease in tigecycline minimum inhibitory concentrations in both Pseudomonas spp and Proteus spp. Our study provides evidence to suggest that tmexCD-toprJ contributes to the antimicrobial resistance phenotypes in different bacterial species. tmexCD-toprJ has disseminated among diverse species of clinical pathogens, which warrants timely monitoring in clinical pathogens. National Natural Science Foundation of China, Guangdong Major Project of Basic and Applied Basic Research, Natural Science Foundation of Jiangsu Province.

Candida parapsilosis Mdr1B and Cdr1B Are Drivers of Mrr1-Mediated Clinical Fluconazole Resistance.

Candida parapsilosis is a common cause of invasive candidiasis worldwide and is the most commonly is7olated Candida species among pediatric and neonatal populations. Previous work has demonstrated that nonsynonymous mutations in the gene encoding the putative transcription factor CpMrr1 can influence fluconazole susceptibility. However, the direct contribution of these mutations and how they influence fluconazole resistance in clinical isolates are poorly understood. We identified 7 nonsynonymous CpMRR1 mutations in 12 isolates from within a collection of 35 fluconazole-resistant clinical isolates. The mutations leading to the A854V, R479K, and I283R substitutions were further examined and found to be activating mutations leading to increased fluconazole resistance. In addition to CpMDR1, we identified two other genes, one encoding a major facilitator superfamily (MFS) transporter (CpMDR1B, CPAR2_603010) and one encoding an ATP-binding cassette (ABC) transporter (CpCDR1B, CPAR2_304370), as being upregulated in isolates carrying CpMRR1-activating mutations. Overexpression of CpMDR1 in a susceptible strain and disruption in resistant clinical isolates that overexpress CpMDR1 had little to no effect on fluconazole susceptibility. Conversely, overexpression of either CpMDR1B or CpCDR1B increased resistance, and disruption in clinical isolates overexpressing these genes decreased fluconazole resistance. Our findings suggest that activating mutations in CpMRR1 represent important genetic determinants of fluconazole resistance in clinical isolates of C. parapsilosis, and unlike what is observed in Candida albicans, this is primarily driven by upregulation of both MFS (CpMdr1B) and ABC (CpCdr1B) transporters.

Water uptake patterns of tropical canopy trees in Borneo: species-specific and temporal variation and relationships with aboveground traits

Root water uptake depth and its temporal variation are important determinants of tree mortality, resource partitioning and drought resistance; however, their effects on tropical trees remain poorly understood. In this study, we investigated interspecific differences in water uptake depth and its temporal variation using stable isotope analysis and examined the relationships between water uptake depth and aboveground traits in a humid aseasonal tropical rainforest in Borneo. Species-specific differences in water uptake depth were examined for six dominant dipterocarp species. Temporal variation in water uptake depth for various canopy trees was assessed in three periods with different soil moisture conditions. We then examined the relationships between water uptake depth and aboveground traits including wood density, maximum tree height, flowering frequency and growth rate. Dipterocarpus globosus appeared to be more reliant on deep water resources than the other dipterocarp species. Water uptake from the soil layers varied among the three sampling periods. Trees generally utilized deeper soil water during the second driest sampling period, when temperatures were lowest. During the driest and wettest sampling periods, species with higher flowering frequencies tended to preferentially uptake deep soil water. These results suggest that low temperature and soil moisture promote increased deep soil water uptake in the study region. Dynamic relationships between water uptake patterns and aboveground tree traits may be related to resource partitioning among co-existing species.

Fibroblast-Induced Paradoxical PI3K Pathway Activation in PTEN-Competent Colorectal Cancer: Implications for Therapeutic PI3K/mTOR Inhibition.

PurposeTumor-microenvironment interactions are important determinants of drug resistance in colorectal cancer (CRC). We, therefore, set out to understand how interactions between genetically characterized CRC cells and stromal fibroblasts might influence response to molecularly targeted inhibitors.TechniquesSensitivity to PI3K/AKT/mTOR pathway inhibitors of CRC cell lines, with known genetic background, was investigated under different culture conditions [serum-free medium, fibroblasts’ conditioned medium (CM), direct co-culture]. Molecular pathway activation was monitored using Western Blot analysis. Immunoprecipitation was used to detect specific mTOR complex activation. Immunofluorescence was used to analyze cellular PTEN distribution, while different mutant PTEN plasmids were used to map the observed function to specific PTEN protein domains.ResultsExposure to fibroblast-CM resulted in increased growth-inhibitory response to double PI3K/mTOR inhibitors in PTEN-competent CRC cell lines harboring KRAS and PI3K mutations. Such functional effect was attributable to fibroblast-CM induced paradoxical PI3K/mTORC1 pathway activation, occurring in the presence of a functional PTEN protein. At a molecular level, fibroblast-CM induced C-tail phosphorylation and cytoplasmic redistribution of the PTEN protein, thereby impairing its lipid phosphatase function and favored the formation of active, RAPTOR-containing, mTORC1 complexes. However, PTEN’s lipid phosphatase function appeared to be dispensable, while complex protein-protein interactions, also involving PTEN/mTOR co-localization and subcellular distribution, were crucial for both mTORC1 activation and sensitivity to double PI3K/mTOR inhibitors.Data InterpretationMicroenvironmental cues, in particular soluble factors produced by stromal fibroblasts, profoundly influence PI3K pathway signaling and functional response to specific inhibitors in CRC cells, depending on their mutational background and PTEN status.

226-OR: Reduced Fatty Acid Oxidation Prevents Intralipid-Induced Hepatic Insulin Resistance in Humans

Mitochondrial handling of excess lipid substrate may be an important determinant of insulin resistance. Animal models support insulin signaling defects resulting from both defective fatty acid oxidation (FAO) with subsequent intra-cellular lipid accumulation on the one hand and enhanced FAO flux on the other. To explore the relationship between FAO and insulin sensitivity, we measured the change in intra-hepatic lipids (IHL) by MRS and hepatic insulin sensitivity during a 5-hour hyperinsulinemic euglycemic clamp with co-infusion of either a control glycerol solution versus a 20% intralipid solution, in random order and separated by 4 months. The study cohort included 18 subjects with an inherited defect in FAO (FAOD; including Carnitine Palmitoyltransferase 2, Long chain 3-Hydroxyacyl-CoA Dehydrogenase, Very Long-chain or Medium-chain Acyl-CoA Dehydrogenase Deficiencies) and 18 matched normal healthy controls. Endogenous glucose production (EGP) before the start of the insulin infusion was similar between FAOD and controls. In controls, insulin suppressed EGP during the glycerol control clamp but this effect was significantly reduced during intralipid infusion (P&amp;lt;0.05, reduced hepatic insulin sensitivity) . In FAOD subjects, insulin suppressed EGP similarly during the glycerol and intralipid clamps (no impairment in insulin action) . Untargeted lipidomics /metabolomics of plasma measured at the end of the intralipid clamp demonstrated significant elevations of plasma acylcarnitines and some triglyceride species but lower production of ketones, and several phospholipids in subjects with FAOD compared to controls. Neither increased IHL nor elevated plasma acylcarnitines in FAOD subjects were associated with hepatic insulin resistance. In fact, impaired FAO protected against intralipid-induced hepatic insulin resistance suggesting that increased FAO flux rather than impaired mitochondrial function is a major determinant of insulin resistance in the liver. Disclosure J.Q.Purnell: Advisory Panel; Novo Nordisk. A.N.Gregor: None. D.Choi: None. E.Baetscher: None. W.Rooney: Consultant; Ultragenix, Research Support; Italofarmco, Revalesio. C.O.Harding: None. M.B.Gillingham: None. Funding NIH/NIDDK: R01DK102813NIH/NCATS: UL1TR002369

Description of a new non-Tn4401 element (NTEKPC-IIe) harboured on IncQ plasmid in Citrobacter werkmanii from recreational coastal water

Here we describe an IncQ1-like plasmid carrying blaKPC-2 in a new non-Tn4401 element found in Citrobacter werkmanii recovered from coastal water. In vitro and in silico approaches were used to assess antimicrobial resistance determinants, as well as blaKPC-2 vicinities. The LB-887 isolate showed a multidrug-resistant phenotype and was identified as C. werkmanii. Resistome analysis identified further acquired resistance determinants to β-lactams, aminoglycosides, sulphonamides/trimethoprim, tetracyclines, chloramphenicol, macrolides, rifampicin and fluoroquinolones. Plasmidome included incompatibility groups IncA, IncC2, IncR, Col and IncQ families. The blaKPC-2 was inserted on a new variant of NTEKPC-II, called here NTEKPC-IIe, carried by an InQ1-like plasmid of 7930 kb (pKPC-LB887). NTEKPC-IIe differed from NTEKPC-IId by the complete absence of ISKpn6-tnpA. The InQ1-like backbone harbouring this element had been described in Enterobacterales recovered from clinical and environmental settings. Unravelling genetic structures related to blaKPC dissemination in different settings may provide clues on the main forces driving evolution of this important resistance determinant. Indeed, the occurrence of blaKPC in a new NTEKPC variant from an environmental source highlights the ongoing evolution of this mobile genetic element. In addition, blaKPC carriage on a small and highly mobilizable IncQ plasmid in C. freundii complex from recreational water, similar to others found in clinical isolates, may suggest its relevance for blaKPC-2 dissemination among different compartments.