Release of large amounts of lipopolysaccharides from Pseudomonas aeruginosa cells reduces their susceptibility to colistin

Pseudomonas aeruginosa is an important etiological agent of opportunistic infections. Injectable colistin is available as a last-line treatment option for multidrug-resistant P. aeruginosa infections. When cells were inoculated at a high number, colistin-susceptible P. aeruginosa grew on agar medium containing colistin at a concentration 10-fold higher than the minimum inhibitory concentration without acquiring colistin resistance. This study examined the responsible mechanism for growth in the presence of a high concentration of colistin. Cell wash fluid derived from P. aeruginosa efficiently reduced colistin antimicrobial activity. This reduction was mediated by lipopolysaccharide (LPS) in the wash fluid. Extracellular LPS inhibited colistin activity more effectively than cell-bound LPS in fixed cells. Cell wash fluids from Escherichia coli and Acinetobacter baumannii also reduced colistin activity; however, they were less potent than those from P. aeruginosa. The amount of LPS in cell wash fluid from P. aeruginosa was approximately 10-fold higher than that in fluid from E. coli or A. baumannii. In conclusion, cell-free LPS derived from bacterial cells inhibited the antimicrobial activity of colistin, and this effect was greatest for P. aeruginosa. Thus, large amounts of broken and dead cells of P. aeruginosa at infection foci will reduce the effectiveness of colistin, even against cells that have not yet acquired resistance.     

Impact of the colistin resistance gene mcr-1 on bacterial fitness

A Klebsiella pneumoniae isolate harbouring a 217 kb IncHI2-type plasmid (pKP2442) encoding the colistin resistance gene mcr-1 was isolated from a leukaemia patient. pKP2442 was mobilised by intragenus and intergenus transconjugation from the clinical isolate to Escherichia coli J53 (transconjugation frequency 6.86?×?10^?8?±?5.57?×?10^?8) and K. pneumoniae PRZ (transconjugation frequency 4.04?×?10^?8?±?3.03?×?10^?8), respectively. Since acquisition of resistance determinants often results in a loss of fitness, the impact of mcr-1 on the fitness of E. coli and K. pneumoniae was investigated. Escherichia coli J53 and K. pneumoniae PRZ transformants harbouring the TOPO expression vector encoding mcr-1 displayed significantly decreased growth rates compared with isogenic parental strains and controls. In contrast, competitive growth experiments revealed equal growth rates between E. coli J53 pKP2442 transconjugants (Tc_pKP2442) and the parental strain, whereas K. pneumoniae PRZ Tc_pKP2442 showed significantly reduced growth rates compared with their parental strain (selection rate constant ?1.62?±?0.49), indicating a decrease in fitness. Infection of A549 human lung epithelial cells with Tc_pKP2442 or mcr-1 transformants and controls revealed equal lactate dehydrogenase activities, indicating no significant impact of mcr-1 on cytotoxicity. Likewise, survival of Galleria mellonella larvae infected with mcr-1-expressing strains and isogenic controls was similar. These data indicate that expression of mcr-1 is able to cause a fitness cost when encoded on expression vectors and that acquisition of natural plasmid-borne mcr-1 does not impair fitness in E. coli J53 but negatively influences growth rates in K. pneumoniae PRZ.     

Isolation of an IncP-1 plasmid harbouring mcr-1 from a chicken isolate of Citrobacter braakii in China

The plasmid-mediated colistin resistance gene mcr-1 has been found worldwide, but the diversity of organisms harbouring this gene is unknown. In this study, 12 colistin-resistant Citrobacter spp. isolates were obtained from diseased or dead chickens in China, and PCR analysis indicated that five were positive for mcr-1. One Citrobacter braakii strain (SCC4) with a multidrug-resistant phenotype was chosen for further analysis. SCC4 was resistant or intermediate-resistant to ten of the tested antibiotics, and the colistin minimum inhibitory concentration (MIC) was >4?µg/mL. A conjugation assay demonstrated successful transfer of colistin resistance to Escherichia coli strain J53 at a frequency of 10^–7 cells per recipient cell. Whole-genome sequencing revealed that SCC4 contained 13 antibiotic resistance genes in its genome, and the mcr-1 gene resided on a 44-kb self-transmissible IncP-type plasmid of a recently discovered IncP-1 clade. In addition, the mcr-1 gene was part of an insertion element (ISApl1–mcr-1–orf–ISApl1) that was excised from the plasmid as a circular intermediate form. This is the first report of mcr-1-posiitve C. braakii of animal origin and these findings highlight the fact that the mcr-1 gene can be found in normal enteric flora as part of broad-host-range plasmids.     

Wide dissemination of SCCfusC in fusidic acid-resistant coagulase-negative staphylococci and implication for its spread to methicillin-resistant staphylococcus aureus in Taiwan

The fusidic acid (FUS) resistance determinants fusB, fusC, fusD and fusF in coagulase-negative staphylococci (CoNS) clinical isolates were examined. Among 208 FUS-resistant isolates, the fusB gene was the most common resistance determinant in each species, except in Staphylococcus hominis subsp. hominis or in species carrying intrinsic fusD or fusF. In S. hominis subsp. hominis, the fusC gene was the major determinant responsible for FUS resistance. To understand the genetic context of fusC in S. hominis subsp. hominis, 31 fusC-positive S. hominis subsp. hominis isolates were examined. Among these isolates, 14 carried SCCfusC, 3 carried an SCC₄₇₆-like element and 7 carried a new SCC structure (SCC₃₃₉₀). As shown by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analyses, the S. hominis subsp. hominis clinical isolates showed limited clonality. Taken together, SCCfusC has been found in S. hominis subsp. hominis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus capitis subsp. ureolyticus and Staphylococcus aureus, suggesting its wide distribution and spread among different species of staphylococci.     

The prevalence of colistin resistance in Escherichia coli and Klebsiella pneumoniae isolated from food animals in China: coexistence of mcr-1 and blaNDM with low fitness cost

Increasing colistin resistance is a global concern because colistin is used as a last resort for the treatment of carbapenem-resistant Enterobacteriaceae infections. The plasmid-mediated colistin resistance gene, mcr-1 was found in distinct bacterial species isolated from humans, animals, and the environment. In this study, farms in four different agricultural provinces in China were investigated to determine the occurrence of the antimicrobial resistance and related genes. A total of 373 Escherichia coli and 54 Klebsiella pneumoniae were isolated from 510 non-duplicated samples. Of the E. coli and K. pneumoniae isolates, 72.7% and 66.7%, respectively, were susceptible to colistin. Isolates resistant to colistin comprised 46.6% of the samples isolated from Shandong, and 17.8% and 16.4% of the samples from Jilin and Henan, respectively. Twenty-six carbapenem-resistant E. coli isolates were resistant to colistin, in which both mcr-1 and bla_NDM were present. Specifically, the co-existence was found in isolates from animals and sewage. Most of the resistance genes were located on plasmids and were 40–244 kilobases. Growth curves of transconjugants carrying mcr-1, bla_NDM-1, bla_NDM-4, bla_NDM-5, and bla_NDM-9 showed a low fitness cost compared with the recipient. In conclusion, mcr-1 was widespread in E. coli and K. pneumoniae isolated from farms in China. Co-existence of mcr-1 and bla_NDM-9 was identified in different sequence types of E. coli with low fitness cost from various origins, indicating an urgent need to take measures for decreasing dissemination.     

慢性U50488H处理对大鼠心脏k－结合特性和生理特性的影响

受体结合分析研究表明，慢性Ｕ５０４８８Ｈ处理后，Ｂｍａｘ无明显改变，而Ｋｄ显著增大，表明ｋ－受体结合位点数量无改变而亲和力降低。在离体灌流心脏，Ｕ５０４８８Ｈ（１０－６ｍｏｌ·Ｌ－１）引起对照组心率、心肌收缩力降低和室性早搏增多；而在慢性Ｕ５０４８８Ｈ处理组，这种效应消失，表明心脏已对Ｕ５０４８８Ｈ产生耐受。以上结果表明，慢性Ｕ５０４８８Ｈ处理导致心脏对该激动剂耐受性的产生，但不伴有相应受体的下调现象。Ｐ＜０．０１但Ｋｄ值比对照组明显增加。２．２Ｕ５０４８８Ｈ对大鼠心脏的作用主要观察心率、心肌收缩力和心脏节律的改变。１０－７ｍｏｌ·Ｌ－１的Ｕ５０４８８Ｈ对这三种参数无明显影响。在３×１０－７ｍｏｌ·Ｌ－１，Ｕ５０４８８Ｈ明显降低心率。１０－６ｍｏｌ·Ｌ－１的Ｕ５０４８８Ｈ降低心率、心肌收缩力并诱发室性早搏。在慢性处理研究中，Ｕ５０４８８Ｈ（１０－６ｍｏｌ·Ｌ－１）对生理盐水处理组与上相似；但其对慢性Ｕ５０４８８Ｈ处理组几乎无任何作用（附表）。３讨论在本研究中，大鼠经慢性注射Ｕ５０４８８Ｈ４ｄ，产生了对Ｕ５０４８８Ｈ的低温效应耐受性，且能取消１０－６ｍｏｌ·Ｌ－１Ｕ５０４８８Ｈ对心率、心肌收缩力和心律的效应，?     

Synergistic microbicidal effect of cationic antimicrobial peptides and teicoplanin against planktonic and biofilm-encased Staphylococcus aureus

Antibiotic resistance and biofilm formation are the main reasons for failure in treatment of bacterial infections. This study aimed to identify synergistic combinations of conventional antibiotics and novel synthetic antimicrobial and antibiofilm peptides (SAAPs) inspired by the structures of the natural human cationic peptides LL-37 and thrombocidin-1 (TC-1). The LL-37-inspired lead peptide SAAP-148 was combined with antibiotics of different classes against Staphylococcus aureus, and showed synergy with teicoplanin. Synergy with teicoplanin was also observed with LL-37, the LL-37-inspired SAAP-276 and the TC-1-inspired TC84. Interestingly, no synergy was observed against Staphylococcus epidermidis. Furthermore, teicoplanin combined with SAAP-148 or SAAP-276 showed strong interaction against S. aureus biofilms. The dltABCD operon and the mprF gene in S. aureus conferred resistance to LL-37, but SAAP-148 proved to be indifferently potent against wild-type, ΔdltA and ΔmprF S. aureus strains. When used alone, relatively high concentrations of both LL-37 and teicoplanin (30–120 µM and 4–32 mg/L, respectively) were required to kill S. aureus. Resistance to LL-37 in S. aureus was overcome by combined use of teicoplanin and LL-37. Thus, teicoplanin potentiates peptide LL-37, enhancing the efficacy of the innate defence, and combining the novel peptides with teicoplanin offers potential for enhanced efficacy of treatment of S. aureus infections, including biofilms.     

Moxifloxacin plus rifampin as an alternative for levofloxacin plus rifampin in the treatment of a prosthetic joint infection with Staphylococcus aureus

Objectives

The combination of a fluoroquinolone with rifampin is one of the cornerstones in the treatment of prosthetic joint infections (PJI) caused by staphylococci. Moxifloxacin is highly active against methicillin–susceptible Staphylococcus aureus (MSSA) and, therefore, is an attractive agent to use. However, several studies reported a lowering in serum moxifloxacin levels when combined with rifampin. The clinical relevance remains unclear. We determined the outcome of patients with early acute PJI caused by MSSA treated with either moxifloxacin/rifampin or levofloxacin/rifampin.

Deoxyribonucleases of the Oriental hornet (Vespa orientalis) venom—II. Partial characterization and effects in vivo on insects and mammals

The presence of DNases in Oriental hornet venom sac extract and venom glands and their activity on invertebrates (bees) and vertebrates (mice and cats) were assessed. It was found that the DNase activity is greatest at neutral pH in the venom sacs and in the acid glands, with very little activity being demonstrated in the alkaline gland. Queen hornets showed greater DNase activity in their acid glands than in their sacs. Workers, on the other hand, showed greater activity in the venom sacs than in the glands. Bees, mice and cats, when injected with hornet venom, showed an increase in nucleic acids, but the onset of the appearance of the DNA degradation products varied with the species: in bees after 5 min, in mice after 7 hr and in cats after 24 hr.     

Synthesis of deuterium- and tritium-labeled 4-hydroxyandrostene-3,17-dione,an aromatase inhibitor,and its metabolism in vitro and in vivo in the rat

The metabolism of the aromatase inhibitor 4-hydroxyandrostenedione (4-OHA) was studied in vitro and in vivo in the rat. To accomplish this, deuterium- and tritium-labeled 4-OHA were prepared from 4-hydroxyandrosta-4,6-diene-3,17-dione. The latter was synthesized from 4-androstene-3,17-dione. Using deuterated 4-OHA in in vitro incubations of rat ovarian microsomes, 4-hydroxytesterone (4-OHT) was identified by gas chromatography/mass spectroscopy as the major metabolite. 4-OHT constituted approximately 20% of the total radioactivity from [6,7-³H]-4-OHA in the ovarian microsomal incubations. Conversion of [6,7-³H]-4-OHA to 4-hydroxyestrone was approximately 0.1%. The major metabolite of [6,7-³H]-4-OHA in vivo identified in the free, neutral fraction of rat blood was 3β-hydroxyandrostane-4,17-dione. This metabolite accounted for approximately 5% of the total radioactivity in the blood, whereas 4-OHT accounted for only 0.1%. 4-OHT inhibited in vitro ovarian aromatization by 59%, but 3β-hydroxyandrostane-4,17-dione had little effect. It was concluded that the in vivo effects of 4-OHA previously reported are largely due to its own activity although additional effects of its metabolic products cannot be excluded.     

Cucurbitacin B induces inhibitory effects via the CIP2A/PP2A/C-KIT signaling axis in t(8;21) acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common subtype of hematological malignancy in humans, and its incidence increases with age. The treatment of AML still faces challenges. Therefore, there is an urgent need to develop more effective targeted therapies. The receptor tyrosine kinase C-KIT confers critical proliferative signals to AML. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an endogenous inhibitor of protein phosphatase 2A (PP2A), which promotes the growth and transformation of various solid tumors. These actions make CIP2A a promising target for tumor treatment. Here, we reported the effects and underlying mechanisms of a natural compound, cucurbitacin B (CuB), on AML. We reported that CuB suppressed growth and induced apoptosis in AML cells. The inhibition of growth and activation of apoptosis were mediated through CuB-induced downregulation of the CIP2A/PP2A/C-KIT signal pathway. Furthermore, CuB inactivated the JAK2 and STAT3 molecules downstream of C-KIT via the downregulation of CIP2A. These results advance our understanding of CuB-induced growth inhibition and apoptosis and support further investigation of CuB as a CIP2A inhibitor for AML therapies.     

Effects of neurohumoral and adrenergic agents on cyclic amp levels in various areas of the rat brain in vitro

When norepinephrine (NE) at 10^?5 M was added to slices of various areas of the rat brain in vitro there was a 2–5 fold increase in the level of adenosine 3',5'-monophosphate (cyclic AMP) within 6 min in all areas except the cerebellum. The basal level of cyclic AMP in the cerebellum was higher than in other areas of the rat brain, and the response to NE was smaller. Histamine and serotonin (5-HT) at 10^?5 M had no effect on cyclic AMP in any brain area. However, 5-HT when added simultaneously with NE, prevented the rise in cyclic AMP elicited by NE in the hypothalamus, midbrain and brainstem. Pargyline elevated the basal levels of cyclic AMP in all brain areas but did not potentiate the action of NE. Chronic reserpine pretreatment in the rat caused an enhanced cyclic AMP response to NE. No effect on cyclic AMP in any brain area was observed following dopamine, amphetamine, or prostaglandins E₁ and E₂. Theophylline slightly reduced cyclic AMP levels. In slices of rat hypogthalamus NE, epinephrine, isoproterenol and α-methyl NE, significantly elevated cyclic AMP levels. Phenylephrine and several “false” transmitters were without effect. Further studies in the hypothalamus revealed that both α and β adrenergic blocking agents effectively antagonized the cyclic AMP response to NE. These results are somewhat difficult to interpret because the precise cellular location of the observed changes are unknown.     

In vitro dissolution and bioavailability study of furosemide nanosuspension prepared using design of experiment (DoE)

Background

Nanotechnology can offer the advantages of increasing solubility and bioavailability of delivering drugs like Furosemide. The aim of the current study is to investigate the in vitro and in vivo performance of furosemide nanosuspensions.

Effects of fluoro-dopamines on dopamine receptors (D1, D2, D3 sites)

The ring-fluorinated compounds, 2-, 5- and 6-fluoro-derivatives of dopamine, were tested on three dopamine-sensitive receptor sites in striatal tissue of rat and calf brain. Although the D₁ site (dopamine-sensitive adenylate cyclase) was stimulated by micromolar concentrations of both dopamine and the fluoro-dopamines, the latter were considerably weaker. The D₂ receptor, as measured by the binding of [³H]spiperone, was occupied by similar concentrations of dopamine and the fluoro-dopamines, with ic₅₀ values ranging from 17,000 to 63,000 nM. The D₃ receptor site, as measured by the highaffinity binding of [³H]dopamine, was also occupied by similar concentrations of these compounds. Since it is primarily the D₂ receptor that is related to various dopaminergic behaviours, the similar potencies of dopamine and the fluoro-dopamines at this receptor site further validate the use of ¹⁸F-compounds to study dopamine function by non-invasive means.     

Electropharmacological effects of intracellular Ca2+ handling modulator caldaret on the heart assessed in the halothane-anesthetized dogs

We analyzed how the enhancement of net sarcoplasmic reticulum (SR) Ca²⁺ uptake may affect cardiac electrophysiological properties in vivo by using caldaret which can decrease SR diastolic Ca²⁺ leak, enhance SR Ca²⁺ reuptake and inhibit reverse-mode Na⁺/Ca²⁺ exchanger. Caldaret in doses of 0.5, 5 and 50 μg/kg was intravenously administered over 10 min to the halothane-anesthetized beagle dogs (n = 5), attaining pharmacologically active plasma concentration. The low and middle doses of caldaret increased the ventricular contraction, which could be explained by its on-target pharmacological activities. The high dose enhanced the sinus automaticity followed by its suppression in addition to the increase of the total peripheral resistance, which may be unfavorable for treating diastolic heart failure. The low and middle doses enhanced the atrioventricular conduction, which may have some potential for predisposing the atria to the onset of atrial fibrillation via an induction of mitral and/or tricuspid regurgitation. The middle and high doses of caldaret prolonged the ventricular effective refractory period without altering the intraventricular conduction or repolarization period, which may prevent the onset of ventricular arrhythmias. Thus, modulation of intracellular Ca²⁺ handling by caldaret can induce not only inotropic effect, but also various electrophysiological actions on the in situ heart.     

Determination of gastric-emptying profiles in the rat: influence of oil structure and volume

A simple non-invasive technique was developed for the determination of the gastric-emptying rate of oils in rats, employing a gamma camera and ^99mTc-sulphur colloid as the oil phase marker. Using this method the gastric emptying of 3 oils, arachis oil, Miglyol 812 and liquid paraffin, was investigated. It was shown that both the oil volume and chemical structure altered the rate of gastric emptying.