Management of multidrug resistant Gram-negative bacilli infections in solid organ transplant recipients: SET/GESITRA-SEIMC/REIPI recommendations

Solid organ transplant (SOT) recipients are especially at risk of developing infections by multidrug resistant (MDR) Gram-negative bacilli (GNB), as they are frequently exposed to antibiotics and the healthcare setting, and are regulary subject to invasive procedures. Nevertheless, no recommendations concerning prevention and treatment are available. A panel of experts revised the available evidence; this document summarizes their recommendations: (1) it is important to characterize the isolate's phenotypic and genotypic resistance profile; (2) overall, donor colonization should not constitute a contraindication to transplantation, although active infected kidney and lung grafts should be avoided; (3) recipient colonization is associated with an increased risk of infection, but is not a contraindication to transplantation; (4) different surgical prophylaxis regimens are not recommended for patients colonized with carbapenem-resistant GNB; (5) timely detection of carriers, contact isolation precautions, hand hygiene compliance and antibiotic control policies are important preventive measures; (6) there is not sufficient data to recommend intestinal decolonization; (7) colonized lung transplant recipients could benefit from prophylactic inhaled antibiotics, specially for Pseudomonas aeruginosa; (8) colonized SOT recipients should receive an empirical treatment which includes active antibiotics, and directed therapy should be adjusted according to susceptibility study results and the severity of the infection.     

碳青霉烯类耐药肠杆菌科细菌碳青霉烯及头孢菌素类抗生素耐药机制

目的研究碳青霉烯类耐药肠杆菌科细菌(CRE)对碳青霉烯及头孢菌素类抗生素耐药机制,为临床治疗CRE感染及医院感染的控制提供分子流行病学依据。方法收集2014—2015年临床送检各类标本分离的细菌,使用Microscan Walkaway 40 plus进行细菌鉴定和药敏试验,对分离的CRE进行常见碳青霉烯酶的编码基因(bla_(NDM-1)及bla_(KPC-2))和超广谱β-内酰胺酶编码基因(bla_(TEM)、bla_(SHV)、bla_(CTX-M-1)-like、bla_(CTX-M-2)-like、bla_(CTX-M-8)-like及bla_(CTX-M-9)-like)的检测,同时检测细菌多重耐药相关的Ⅰ类整合子编码基因bla_(inT-1)。结果 2014—2015年共分离7株CRE,检出率为0.30%,1~6号菌株为肺炎克雷伯菌,7号菌株为弗劳地柠檬酸杆菌,7株菌株对β-内酰胺酶抑制剂复合制剂、头孢菌素类、头霉素类、碳青霉烯类抗生素均耐药,3株菌对阿米卡星及四环素敏感,2株菌对复方磺胺甲口恶唑敏感。3株菌检出bla_(NDM-1),2株检出bla_(KPC-2),5株检出bla_(TEM),7株均可检出bla_(SHV),1株检出bla_(CTX-M-1)-like,4株检出bla_(CTX-M-9)-like,5株检出bla_(inT-1),未检出基因bla_(CTX-M-2)-like及bla_(CTX-M-8)-like。结论 bla_(NDM-1)及bla_(KPC-2)是导致7株CRE对碳青霉烯类抗生素耐药的重要机制,bla_(TEM)、bla_(SHV)及bla_(CTX-M-9)-like是导致对头孢菌素类抗生素耐药的主要原因,bla_(inT-1)在CRE多重耐药及耐药基因传播中发挥着巨大的作用。     

Temporal and spatial regulation of CRE recombinase expression in gonadotrophin-releasing hormone neurones in the mouse

Gonadotrophin-releasing hormone (GnRH) neurones located within the brain are the final neuroendocrine output regulating the reproductive hormone axis. Their small number and scattered distribution in the hypothalamus make them particularly difficult to study in vivo . The Cre/loxP system is a valuable tool to delete genes in specific cells and tissues. We report the production of two mouse lines that express the CRE bacteriophage recombinase in a GnRH-specific manner. The first line, the GnRH-CRE mouse, contains a transgene in which CRE is under the control of the murine GnRH promoter and targets CRE expression specifically to GnRH neurones in the hypothalamus. The second line, the GnRH-CRETeR mouse, uses the same murine GnRH promoter to target CRE expression to GnRH neurones, but is modified to be constitutively repressed by a tetracycline repressor (TetR) expressed from a downstream tetracycline repressor gene engineered within the transgene. GnRH neurone-specific CRE expression can therefore be induced by treatment with doxycycline which relieves repression by TetR. These GnRH-CRE and GnRH-CRETeR mice can be used to study the function of genes expressed specifically in GnRH neurones. The GnRH-CRETeR mouse can be used to study genes that may have distinct roles in reproductive physiology during the various developmental stages.     

Involvement of basic amino acid residues in transmembrane regions 6 and 7 in agonist and antagonist recognition of the human platelet P2Y(12)-receptor

The P2Y(12)-receptor plays a prominent role in ADP-induced platelet aggregation. In the present study, we searched for amino acid residues involved in ligand recognition of the human P2Y(12)-receptor. Wild-type or mutated receptors were expressed in 1321N1 astrocytoma cells and Chinese hamster ovary (CHO) cells. There were no major differences in cellular expression of the constructs. Cellular cAMP production and cAMP response element (CRE)-dependent luciferase expression was increased by isoproterenol (astrocytoma cells) or forskolin (CHO cells). In cells expressing wild-type receptors, R256K or S101A mutant constructs, 2-methylthio-ADP inhibited the induced cAMP production with IC(50) concentrations of about 0.3nM. In cells expressing R256A constructs, the IC(50) concentration amounted to 25nM. In cells expressing H253A/R256A, Y259D and K280A constructs, 2-methylthio-ADP failed to affect the cellular cAMP production. Moreover, in cells expressing Y259D and K280A constructs, 2-methylthio-ADP did also not change the forskolin-induced CRE-dependent luciferase expression and caused only small increases in the serum response element-dependent luciferase expression. The antagonist cangrelor had similar potencies at wild-type receptors and R256A constructs (apparent pK(B)-value at wild-type receptors: 9.2). In contrast, reactive blue-2 had a lower potency at the R256A construct (apparent pK(B)-value at wild-type receptors: 7.6). In summary, the data indicate the involvement of Arg256, Tyr259 and, possibly, H253 (transmembrane region TM6) as well as Lys280 (TM7) in the function of the human P2Y(12)-receptor. Arg256 appears to play a role in the recognition of nucleotide agonists and the non-nucleotide antagonist reactive blue-2, but no role in the recognition of the nucleotide antagonist cangrelor.     

Use of chimeric melanocortin-2 and -4 receptors to identify regions responsible for ligand specificity and dependence on melanocortin 2 receptor accessory protein

The melanocortin 2 (MC₂) receptor differs from other melanocortin family members in its pharmacological profile and reliance on an accessory protein, MC₂ receptor accessory protein (MRAP), for surface expression and signal transduction. To identify features of the MC₂ receptor responsible for these characteristics, we created chimeras between MC₂ and MC₄ receptors and expressed these in CHO cells, where MRAP is essential for trafficking and signaling by MC₂ but not MC₄ receptors. Replacing the first transmembrane segment of the MC₂ receptor with the corresponding region from the MC₄ receptor allowed some surface expression in the absence of an accessory protein, while ACTH-induced cAMP production remained entirely MRAP-dependent. On the other hand, replacing the last two transmembrane domains, third extracellular loop and C-terminal tail of the MC₄ receptor with the corresponding regions from the MC₂ receptor resulted in MRAP-dependent signaling. Surprisingly, replacing the second and third transmembrane domains and the intervening first extracellular loop of MC₂ receptors with MC₄ sequences generated a chimera (2C2) that responded to both adrenocorticotropic hormone (ACTH) and to the potent MSH analog 4-norleucine-7-d-phenylalanine-α-melanocyte stimulating hormone (NDP-α-MSH), which does not activate native MC₂ receptors. The 2C2 chimeric receptor was able to respond to NDP-α-MSH without MRAP, but MRAP shifted the EC50 value for NDP-α-MSH to the left and caused constitutive activity. These results identify the first transmembrane domain as important for surface expression and regions from the second to third transmembrane segments of the MC₂ receptor as important for MRAP dependent-signal transduction and ligand specificity.