RESUMO
BACKGROUND: Ventilator-associated pneumonia (VAP) is a common nosocomial infection associated with high morbidity due to multidrug-resistant (MDR) pathogens. The purpose of this study was to determine the occurrence of extended-spectrum ß-lactamase (ESBL) genes, especially bla CTX-M-15, in Klebsiella pneumoniae (K. pneumoniae)-associated VAP and to investigate the antimicrobial resistance patterns and molecular epidemiological characteristics of K. pneumoniae strains. MATERIALS AND METHODS: From January 2013 to December 2015, we retrospectively collected 89 VAP-causing K. pneumoniae isolates from tertiary-care hospitals in China, among which ESBL-producing strains were assessed for antimicrobial susceptibility. Several antibiotic resistance genes of clinical relevance in K. pneumonia isolates producing ESBL were investigated. Polymerase chain reaction (PCR) and DNA sequencing were employed to characterize the genetic contexts of bla CTX-M-15. Conjugative plasmids carrying bla CTX-M-15 were obtained by mating and further subjected to replicon typing. The genetic relatedness of isolates was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. RESULTS: All of the 30 ESBL-producing isolates identified displayed MDR phenotype, with bla SHV, bla CTX-M, bla OXA, and bla TEM detected in 21, 21, 1, and 20 isolates, respectively. bla CTX-M-15 was the most prevalent ESBL gene (19/30, 63.33%), and ISEcp1 was detected 48 bp upstream of 15 bla CTX-M-15 genes. Based on S1-PFGE analyses, 25 isolates exhibited different plasmid profiles, ranging from ~70 to 320 kb. The bla CTX-M-15 with bla TEM and qnr genes and the ISEcp1 element from eight isolates were co-transferrable to recipients via conjugation, with IncFIB, IncFIC, and IncFII being the most prevalent replicons. Twenty different PFGE patterns and 11 sequence types were identified, with ST304 being dominant. CONCLUSION: This work reports the emergence of bla CTX-M-15 in K. pneumoniae-induced VAP in China. We showed that IncFIB, IncFIC, and/or IncFII plasmids carrying bla CTX-M-15 with bla TEM, qnr resistance genes, and the ISEcp1 element mediate the local prevalence in K. pneumoniae-associated VAP.
RESUMO
Schwann cell apoptosis is one of the characteristics of diabetic peripheral neuropathy (DPN). The mammalian target of rapamycin (mTOR) is a multifunctional signaling pathway that regulates cell apoptosis in various types of tissues and cells. To investigate whether the mTOR pathway is involved in cell apoptosis in the Schwann cells of DPN, diabetic mice and rat Schwann cells (RSC96) were chosen to detect phospho-mTOR (Ser 2448), phospho-S6K1 (Thr 389), phospho-4EBP1 (Thr 37/46), Bcl-2, Bax and cleaved caspase-3 by diverse pathological and biological techniques. The results showed that phospho-mTOR (Ser 2448) was decreased in the sciatic nerves of diabetic mice, concomitant with decreased Bcl-2, increased Bax, cleaved caspase-3 and cell apoptosis. In addition, high glucose treatment for 72â¯h caused a 35.95% decrease in the phospho-mTOR (Ser 2448)/mTOR ratio, a 65.50% decrease in the phospho-S6K1 (Thr 389)/S6K1 ratio, a 3.67-fold increase in the Bax/Bcl-2 ratio and a 1.47-fold increase in the cleaved caspase-3/caspase-3 ratio. Furthermore, mTORC1 inhibition, rather than mTORC2 inhibition, resulted in mitochondrial controlled apoptosis in RSC96 cells by silencing RAPTOR or RICTOR. Again, suppression of the mTORC1 pathway by a chemical inhibitor led to mitochondrial controlled apoptosis in cultured RSC96 cells in vitro. By contrast, activation of the mTORC1 pathway with MHY1485 prevented decreased phospho-S6K1 (Thr 389) levels caused by high glucose and cell apoptosis. Additionally, constitutive activation of S6K1 avoided high glucose-induced cell apoptosis in RSC96 cells. In summary, our findings suggest that activating mTORC1/S6K1 signaling in Schwann cells may be a promising strategy for the prevention and treatment of DPN.
Assuntos
Apoptose , Nefropatias Diabéticas/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Células de Schwann/metabolismo , Animais , Caspase 3/metabolismo , Linhagem Celular , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/patologia , Glucose/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos , Mitocôndrias/efeitos dos fármacos , Naftiridinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/antagonistas & inibidores , Ratos , Proteína Regulatória Associada a mTOR/antagonistas & inibidores , Células de Schwann/efeitos dos fármacos , Células de Schwann/enzimologia , Nervo Isquiático/enzimologia , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Serious infections in intensive care unit patients caused by multidrug-resistant (MDR) Klebsiella pneumoniae represent a major threat worldwide owing to increased mortality and limited treatment options. With the application of tigecycline for MDR pathogens, tigecycline-non-susceptible K. pneumoniae isolates have recently emerged in China. To identify the susceptibility profile of MDR K. pneumoniae to tigecycline and evaluate the molecular characterization of tigecycline resistance, 214 MDR K. pneumoniae isolates were collected from blood samples of patients in intensive care units. MICs and clonal relatedness were determined by standard broth microdilution and multilocus sequence typing, respectively. Expression levels of efflux pumps and their global regulators were examined using real-time PCR. Mutations of local repressor were identified by PCR and sequencing. Our results show that the tigecycline resistance rate of 214 MDR K. pneumoniae isolates was 6.07 %. ST11 was the predominant clone type of tigecycline-non-susceptible K. pneumoniae isolates. Expression of efflux pump AcrB and global regulator RamA correlated with tigecycline MICs (AcrB: x2=8.91, P=0.03; RamA: x2=13.91, P<0.01), and mean expression levels of AcrB for the MICs ≥4 mg l-1 were significantly higher than MICs ≤2 mg l-1 (t=2.48, P=0.029). In addition, one tigecycline-resistant isolate harboured a deletion mutation in the ramR gene. These data indicated a linear correlative trend for overexpression of the AcrB and the tigecycline MICs resulting from the upregulation of RamA. The emergence of molecular type ST11 of MDR K. pneumoniae isolates should be monitored to identify factors that contribute to tigecycline resistance in intensive care units.