A high-throughput sequencing determination method for upstream genetic structure (UGS) of ISEcp1-blaCTX-M transposition unit and application of the UGS to classification of bacterial isolates possessing blaCTX-M

IntroductionBecause bla_CTX-M is responsible for resistance of bacteria to the third generation cephalosporins, location of bla_CTX-M could be a good indicator for classifying bacterial isolates harboring bla_CTX-M in molecular epidemiology. However, determination of bla_CTX-M location has been difficult when multiple copies of ISEcp1 were found on bacterial genome. We aimed to establish a high-throughput analytical method for upstream genetic structures (UGS) of ISEcp1 to facilitate determination of bla_CTX-M location.MethodsExtracted DNA samples obtained from 168 Escherichia coli isolates possessing bla_CTX-M were digested by restriction enzyme, HaeIII, and the digested DNA fragments were ligated with homemade barcode adaptors. Then, DNA fragments containing UGS of ISEcp1 were amplified and subjected to the Nanopore sequencer.ResultsNucleotide sequences and locations of 168 UGSs obtained from the examined E. coli isolates were determined. Among the 168 determined UGSs, 150 (89.3%) UGS were confirmed on plasmid and classified into eight types. Interestingly, coding sequence of ISEcp1 transposase gene in seven of the eight types were disrupted by IS26 insertion. The remaining 18 (10.7%) UGSs were observed in identical chromosomal region. The obtained nucleotide sequences the locations of UGSs were confirmed by conventional capillary sequencer and Southern blotting, respectively, and any discrepant result was not observed with these confirmation procedures.ConclusionsOur results indicated that the established method was efficient for simultaneously determining at least 100 different UGS, and suggested that the determined UGSs of ISEcp1-bla_CTX-M transposition unit was useful for classification of bacterial isolates harboring bla_CTX-M.     

Simultaneous quantification of plasma levels of 12 antimicrobial agents including carbapenem,anti-methicillin-resistant Staphylococcus aureus agent,quinolone and azole used in intensive care unit using UHPLC-MS/MS method

ObjectivesCritically ill patients in intensive care unit (ICU) are susceptible to infectious diseases, thus empirical therapy is recommended. However, the therapeutic effect in ICU patients is difficult to predict due to fluctuation in pharmacokinetics because of various factors. This problem can be solved by developing personalized medicine through therapeutic drug monitoring. However, when different measurement systems are used for various drugs, measurements are complicated and time consuming in clinical practice. In this study, we aimed to develop an assay using ultra-high performance liquid chromatography coupled with tandem mass spectrometry for simultaneous quantification of 12 antimicrobial agents commonly used in ICU: doripenem, meropenem, linezolid, tedizolid, daptomycin, ciprofloxacin, levofloxacin, pazufloxacin, fluconazole, voriconazole, voriconazole N-oxide which is a major metabolite of voriconazole, and posaconazole.Design & methodsPlasma protein was precipitated by adding acetonitrile and 50% MeOH containing standard and labeled IS. The analytes were separated with an ACQUITY UHPLC CSH C18 column, under a gradient mobile phase consisting of water and acetonitrile containing 0.1% formic acid and 2 mM ammonium formate.ResultsThe method fulfilled the criteria of US Food and Drug Administration for assay validation. The recovery rate was more than 84.8%. Matrix effect ranged from 79.1% to 119.3%. All the calibration curves showed good linearity (back calculation of calibrators: relative error ≤ 15%) over wide concentration ranges, which allowed determination of C_max and C_trough. Clinical applicability of the novel method was confirmed.ConclusionsWe have developed an assay for simultaneous quantification of 12 antimicrobial agents using a small sample volume of 50 μL with a short assay time of 7 min. Our novel method may contribute to simultaneous calculation of pharmacokinetic and pharmacodynamic parameters.     

常见抗生素与新型抗菌药物在临床上的研究应用进展

摘要：抗生素的研发与使用有效杀灭和抑制了众多病原菌，挽救了无数人的生命，但由于抗生素的不合理使用，导致细菌耐 药不断出现，耐药菌的感染已严重地威胁人类的生命健康。为此，本文主要对近年来临床上常用的抗生素如β-内酰胺类、喹诺酮 类、氨基糖苷类药物以及新型抗菌药物如抗菌肽和纳米颗粒的应用研究进行总结分析与探讨，为临床上治疗疾病提供一些参考。     

Comparative analysis of different methods used for molecular characterization of Burkholderia cepacia complex isolated from noncystic fibrosis conditions

PurposeBurkholderia is a Gram-negative opportunistic bacterium capable of causing severe nosocomial infections. The aim of this study was to characterize Burkholderia cepacia complex and to compare different molecular methods used in its characterization.MethodsIn this study, 45 isolates of Burkholderia cepacia complex (Bcc) isolated from clinical cases were subjected to RAPD (Random amplified polymorphic DNA), recA-RFLP (Restriction fragment length polymorphism), 16SrDNA-RFLP, whole-cell protein analysis, recA DNA sequencing and biofilm assay.ResultsOf the 45 isolates tested, 97.7% were sensitive to ceftazidime, 82.2% were sensitive to Cotrimoxazole, 73.3% were sensitive to meropenem, 55.5% were sensitive to minocycline and 42.2% were sensitive to levofloxacin. Majority of the isolates harbored all the tested virulence genes except bpeA and cblA. The RAPD generated 11 groups (R1-R11), recA-RFLP 10 groups (A1-A10), 16SrRNA-RFLP 5 groups (S1–S5) and SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis) whole cell protein analysis revealed 12 groups (C1–C12). recA sequencing revealed that most of the isolates belonging to the genomovar III Burkholderia cenocepacia. Though all the methods are found to be efficient in differentiating Burkholderia spp., recA-RFLP was highly discriminatory at 96% similarity value. The study also identified a new strain Burkholderia pseudomultivorans for the first time in the country. Further, recA sequencing could identify the strains to species level. Majority of the multidrug-resistant strains also showed moderate to strong biofilm-forming ability, which further contributes to the virulence characteristics of the pathogens.ConclusionsThe study highlights the importance of combination of molecular methods to characterize Burkholderia cepacia complex. Molecular typing of these human pathogens yields important information for the clinicians in order to initiate the most appropriate therapy in the case of severe infections and to implement preventive measures for the effective control of transmission of Burkholderia spp.     

Clinical isolates of Escherichia coli solely resistant to mecillinam: prevalence and epidemiology

In routine susceptibility testing of Gram-negative bacteria, a particular resistance phenotype was observed: an Escherichia coli isolate from a urine sample exhibited resistance solely to mecillinam (MEC) but was fully susceptible to other β-lactam antibiotics (MEC-R-BL-S). The objectives as this study were to determine the prevalence of this phenotype and to describe the phenotype, molecular epidemiology and genetic background. Between 1 January 2014 and 31 January 2016, MEC-R-BL-S E. coli isolates from urine were collected and genes previously reported as mostly involved in MEC resistance were analysed. The genetic relatedness among isolates was investigated by repetitive element sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST). Ten MEC-R-BL-S isolates were collected, accounting for 0.4% (10/2547) of all E. coli obtained from urine samples, 0.9% (10/1135) of ampicillin-susceptible E. coli isolates and 9.6% (10/104) of MEC-R E. coli isolates. The isolates appeared as small colonies with round morphology and had impaired fitness. The isolates were not clonal and belonged to various extraintestinal or commensal E. coli phylogroups. Mutations in the cysB gene were evidenced in all clinical isolates. In conclusion, microbiologists should be aware of these isolates with a particular susceptibility phenotype, which is not due to error in disk diffusion but is a real non-enzymatic antibiotic resistance pattern.     

Purification and characterisation of a novel antistaphylococcal peptide (ASP-1) from Bacillus sp. URID 12.1

A strong antistaphylococcal peptide (ASP-1) from Bacillus subtilis URID 12.1 strain that is active against cefoxitin- and methicillin-resistant Staphylococcus aureus clinical isolates was purified to homogeneity by solvent extraction, silica gel-based adsorption chromatography and reversed-phase high-performance liquid chromatography. The peptide sequence of ASP-1 as determined by MALDI-TOF/MS and ESI-FTICR-MS was acetylated Phe-Thr-Ala-Val-Dhb-Phe-Ile/Leu. The peptide was further analysed by alkaline hydrolysis, ESI-Q-TOF-MS and an ion mobility assay, which detected the presence of a lactone ring in the intact peptide and a cyclic nature, subsequently revealing the linearised peptide sequence as acPhe-Leu-Phe-Thr-Val-Ala-Dhb. Based on the molecular mass (804.5 Da), peptide sequence and amino acid composition, ASP-1 was identified as a lactone ring-containing peptide similar to TL-119, a poorly studied cyclic depsipeptide. Circular dichroism spectroscopy revealed its predominantly random structure in aqueous solution and its β-sheet conformation in methanol. Minimum inhibitory concentrations (MICs) of the purified peptide against S. aureus and methicillin-resistant S. aureus (MRSA) ranged from 2?µg/mL to 64?µg/mL. At sub-MICs and 1× MIC, ASP-1 showed a strong antibiofilm characteristic. ASP-1 at a concentration of 128?µg/mL did not show haemolytic activity, and no cytotoxicity was observed against hepatic carcinoma and breast carcinoma cell lines at the same concentration. Peptide ASP-1 with anti-MRSA and antibiofilm abilities and non-haemolytic and non-cytotoxic properties has not been reported previously. These findings suggest that it may serve as a lead molecule for developing alternative topical antibacterial agents.     

基于抗菌药物分级管理的我院抗菌药物医嘱评价

摘 要 目的：明确我院三级抗菌药物（非限制使用级、限制使用级、特殊使用级）不合理使用情况的特点及差异,为临床规范使用抗菌药物提供理论依据。 方法：对我院736份出院病历的1 228条抗菌药物医嘱逐条进行合理性点评,并对三级抗菌药物医嘱不合理使用率、分布特点、类型进行比较。 结果：我院三级抗菌药物不合理使用率差异不全相同,以限制使用级为最高（25.0%）,特殊使用级最低（9.8%）,两者比较差异有统计学意义（P<0.01）。以头孢菌素为主的β内酰胺类抗菌药物的不合理医嘱最多。三级抗菌药物不合理用药类型差异有统计学意义（P<0.05）。“抗菌药物用法用量不适宜”、“围手术期预防性使用抗菌药物不合理”为非限制使用级及限制使用级抗菌药物的主要不合理医嘱构成。而“遴选抗菌药物不适宜”则为特殊使用级抗菌药物的主要不合理医嘱构成。结论：我院抗菌药物不合理使用情况较普遍。医院需针对各级抗菌药物使用中存在的主要问题加强抗菌药物的管理。