Persistent drug resistance to tyrosine kinase inhibitors (TKI) has become a hurdle in extending the survival of patients with clear cell renal cell carcinoma (ccRCC). Through microbiome screening of patient samples from a ccRCC cohort treated with the TKI pazopanib, we identified Streptococcus pneumoniae as the dominant intratumoral microbiota in pazopanib-resistant ccRCC samples. Further investigation revealed that S. pneumoniae reprogrammed lipid metabolism in ccRCC cells by depleting manganese (Mn2+) from the tumor microenvironment, consequently facilitating malignant progression and development of pazopanib resistance. S. pneumoniae suppressed S-nitrosylation of tripartite motif-containing protein 28 (TRIM28) by diminishing Mn2+ levels, allowing TRIM28 to physically interact with the transcription factor SP1 to promote the transcription of solute carrier family 27 member 1 (SLC27A1) and lipid deposition. Taken together, these findings indicate that tumor-resident S. pneumoniae plays an important role in conferring pazopanib resistance, suggesting that S. pneumoniae could serve as a potential biomarker of pazopanib response in ccRCC.Significance: Streptococcus pneumoniae is an intratumoral bacteria that drives pazopanib resistance in renal cell carcinoma, highlighting the potential of targeting the tumor microbiome as a strategy to improve treatment outcomes in cancer patients.

Objective:In this study, we constructed ampG knock-out and knock-in strains from a clinically isolated Kp1strain carrying ampR-ampC in its plasmid and compared them with the Kp NTUH-K2044 strain to investigate the relationship between ampG and ampR-ampC-induced expression.Methods:We created the ampG gene deletion mutant strains Kp1-ΔampG and Kp NTUH-K2044-ΔampG with pKO3-km plasmid using homologous recombination technology. We constructed the Kp NTUH-K2044-RC and Kp NTUH-K2044-ΔampG-RC drug resistance model strains with plasmid pACYC184. We constructed the ampG knock-in strains by introducing the ampG genes of Kp1, Enterobacter cloacae 029M, Pseudomonas aeruginosa PAO1, Escherichia coli ATCC25922, and Salmonella typhimurium LT2 into the ampG gene-deleted strains with carrier pet-30a. Real-time polymerase chain reaction (real-time PCR) was used to detect the relative expressions of ampC and ampG mRNAs.Results:Compared with Kp1, the induction phenotype of the ampC of Kp1-ΔampG strain disappeared, the ampC expression was reduced, and the minimal inhibitory concentration (MIC) values of cefoxitin and ceftazidime significant decrease from 128 μg/mL to 1 μg/mL. Based on Kp1, five strain were successfully constructed to complement the ampG genes from five knock-in strain, and all of the above complemented strains showed inducible expression of ampC and restored the expression of ampG to varying degrees, as well as restored resistance to the antimicrobial drugs cefoxitin and ceftazidime (P < 0.05). The ampC and ampG genes were barely expressed in Kp NTUH-K2044-ΔampG-RC when compared with Kp NTUH-K2044-RC. The expressions of ampG and ampC in each knock-in strain were recovered, the induction phenotype of ampC was restored, and the MIC values of cefoxitin and ceftazidime were increased. (P < 0.05).Conclusion:In this study, we found that ampG was an essential regulator for the plasmid-mediated ampC-induced expression in K. pneumoniae.

高毒力肺炎克雷伯菌(hvKP)已成为全球关注的病原菌,其毒力强,更易引起社区感染,患者感染hvKP后预后差、病死率高.令人担忧的是,高耐药的hvKP已经出现,对临床抗感染治疗提出了新的挑战.本文综述了hvKP的临床鉴定和主要的毒力因子,以及耐药菌株和高毒力菌株的重叠表现.

BACKGROUND:Escherichia coli are mostly commensals but also contain pathogenic lineages. It is largely unclear whether the commensal E. coli as the potential origins of pathogenic lineages may consist of monophyletic or polyphyletic populations, elucidation of which is expected to lead to novel insights into the associations of E. coli diversity with human health and diseases.METHODS:Using genomic sequencing and pulsed field gel electrophoresis (PFGE) techniques, we analyzed E. coli from the intestinal microbiota of three groups of healthy individuals, including preschool children, university students, and seniors of a longevity village, as well as colorectal cancer (CRC) patients, to probe the commensal E. coli populations for their diversity.RESULTS:We delineated the 2280 fresh E. coli isolates from 185 subjects into distinct genome types (genotypes) by PFGE. The genomic diversity of the sampled E. coli populations was so high that a given subject may have multiple genotypes of E. coli, with the general diversity within a host going up from preschool children through university students to seniors. Compared to the healthy subjects, the CRC patients had the lowest diversity level among their E. coli isolates. Notably, E. coli isolates from CRC patients could suppress the growth of E. coli bacteria isolated from healthy controls under nutrient-limited culture conditions.CONCLUSIONS:The coexistence of multiple E. coli lineages in a host may help create and maintain a microbial environment that is beneficial to the host. As such, the low diversity of E. coli bacteria may be associated with unhealthy microenvironment in the intestine and hence facilitate the pathogenesis of diseases such as CRC.

目的 分析我院近4年临床分离常见革兰阴性杆菌的临床分布及耐药性特点,为感染相关疾病的早期治疗以及预防院内感染提供指导依据.方法 收集我院2014 ～2017年各临床标本的分离株,法国生物梅里埃公司微生物菌株鉴定试条鉴定菌株,采用纸片扩散法进行药敏试验,采用WHONET5.6软件统计数据分析.结果 我院从临床标本中的得到革兰阴性杆菌共20 270株,其中大肠埃希菌检出率最高,其次为肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌、阴沟肠杆菌等.各分离菌主要集中在ICU,标本主要来源于呼吸道标本.大肠埃希菌对β-内酰胺类药物耐药率较高,而含酶抑制剂类的耐药率较低;肺炎克雷伯菌大多抗菌药物的耐药率略低于大肠埃希菌;鲍曼不动杆菌除了对替加环素的耐药率较低,对于其他抗菌药物耐药率高于60.00％;铜绿假单胞菌对部分抗菌药物的耐药率逐年降低;大肠埃希菌产ESBLs菌株检出率每年均稳定,肺炎克雷伯菌产ESBLs菌株检出率有所上升;CRE检出率较低,却有上升趋势;CRAb检出率每年均在70.00％左右,CRPa检出率有所下降.结论 通过对细菌耐药性进行长期监测,可以有效地控制和预防耐药菌的出现和传播,预防院内感染,指导临床合理用药.

在国家“一带一路”战略背景下,留学生尤其是俄罗斯留学生规模不断扩大.深入了解留学生学习习惯与特点,不断提高其实验诊断学实践操作能力仍是该领域教育工作者新时期面临的巨大挑战.本文介绍了哈尔滨医科大学近年针对留学生实验诊断学实践操作培养的经验,对新时代留学生教学进行了思考.从“学”与“教”两个方面的反馈来看,本次实践技能教学活动达到该校对于临床本科层次的教学要求.

Macrolide streptogramin type B resistance (the MSB phenotype) is a multidrug resistance phenotype in Staphylococcus aureus conferred by the resistance gene msrA. However, bacteria having the MSB phenotype are susceptible to lincosamides and 16-membered ring macrolides, which makes profiling resistance genes necessary and urgent for timely and appropriate use of antimicrobials. In this study, the loop-mediated isothermal amplification (LAMP) assay was optimized for prompt detection of the msrA gene. msrA gene sequences were obtained from the National Center for Biotechnology Information (NCBI) database and primers were designed using the LAMP primer designing software PrimerExplorer v.4, which together recognize seven distinct regions of the msrA gene. The specific LAMP primer set designed in this study could amplify the msrA gene within 25 min at an isothermal temperature of 62 degrees C. More importantly, the msrA gene could be detected at a sensitivity as low as 100 pg. Furthermore, this optimized LAMP assay provided swift detection of the msrA gene even directly from human specimens. In conclusion, this assay may have great clinical application potential for detection of the msrA gene. (C) 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

鲍曼不动杆菌(Acinetobacter baumanii,Ab)为不动杆菌属中最常见的一种革兰阴性杆菌.临床检出率呈上升趋势,耐药率也不断上升.近年来对碳青霉烯类抗生素尤其是亚胺培南耐药的鲍曼不动杆菌日益增多,呈暴发流行.鲍曼不动杆菌外膜蛋白对此类抗生素的低渗透性参与了其耐药机制形成.本文就亚胺培南耐药鲍曼不动杆菌的外膜蛋白与耐药性之间的关系做一综述.

Objective to understand presence of ADC type AmpC enzyme gene in multidrug resistance baumanii and its upstream insertion sequence ISaba1﹒ Method collect 146 strains baumanii separated from specimens of hospitalized patients in Harbin Medical University the Second Affiliated Hospital from October 2011 to June 2012. Test its sensitivity for 11 antibiotics by K-B disk diffusion method﹒ Analyze gene of ADC type AmpC enzyme gene fragments, whole gene of ISaba1-AmpCand ADC-AmpC by PCR and DNA sequencing. Test relative expression of bla ampC gene by fluorescence quantitative PCR method. Result among 146 strains of baumanii, cefoperazone/sulbactam has higher sensitivity (52﹒7%), and other antibiotics has lower sensitivity(P<10﹒0%)﹒ Positive rate of ADC gene was 91﹒7% (134/146), positive rate of AmpC enzyme upstream insertion sequence ISaba1 was 73﹒2% (107/146)﹒ Subtype ADC gene in our hospital was ADC-30 (GU591985 in Genbank)﹒Conclusion multidrug resistance of this group is serious, baumanii has close relationship withβ-lactam antibiotics and ADC AmpC enzyme caused by it, and its upstream insertion sequence ISaba1 has self promoter, which may cause expression increase of AmpC enzyme gene mRNA﹒

Objective To investigate the situation of Pseudomonas aeruginosa strains carrying metallo-β-lactamases and inte-grases in the Second Affiliated Hospital of Harbin Medical University.Methods The phenotype of metallo-β-lactamases were detected by modified Hodge test,double-disc synergy and combination paper method,respectvily.PCR method was used to detecte metallo-β-lactamases genotypes and the integrationⅠ,Ⅱ and Ⅲ.The PCR products of the whole length bla-IMP gene were purified,sequenced and analyzed by Blast.Results Among 62 Pseudomonas aeruginosa strains,metallo-β-lactamases phenotype of 3 by modified hodge test,4 by double-disc synergy test,4 by combination paper method and were all positive 4 of IMP-1 type metallo-β-lactamases of Pseudomonasaeruginosa strains (10%,4/40)were positives by PCR.Inte-grase Ⅰ of 16(40 %,16/40)strains were positives by PCR method in IMP-resistant Pseudomonasaeruginosa,and integraseⅠ of 22.73 %(5/22)were positives in IMP-sensitive Pseudomonasaeruginosa.No other metallo-β-lactamases,integraseⅡandⅢ were detected in this study.Conclusion IMP-type metallo-β-lactamase existed in IMP-resistant Pseudomonasaerugi-nosa isolates of the Second Affiliated Hospital of Harbin Medical University.Most strains carried integraseⅠ,and other re-sistance mechanisms may be associated with multi-drug resistance,so it is important to prevent the Pseudomonasaeruginosa strains which carried metallo-β-lactamases and integrons widely spread in the hospital.

不动杆菌属（Acinetobacter spp.）在临床的检出率日益增高,特别是鲍曼不动杆菌（Acinetobacter baumannii）,耐药性日趋严重,出现了多重耐药（multiple-drug resistance,MDR）,广泛耐药（extensively drug resistance,XDR）以及泛耐药（pan-drug resistance,PDR）的菌株。不动杆菌耐药性复杂,由多种耐药机制参与,而药物主动外排系统的过度表达是主要耐药机制之一,其中耐药结节细胞分化（resistance-nodulation-cell-division,RND）家族,具有广泛的底物外排性,与不动杆菌多重耐药性密切相关,因此研究RND外排泵耐药及其表达调控机制对防治临床不动杆菌感染有一定的理论和现实意义。本文就对不动杆菌RND外排泵的结构特点、耐药性等方面进行详细综述。

鲍曼不动杆菌(Acinetobacter baumannii,Ab)是条件致病菌,易引起医院感染,由于广谱抗菌药物的广泛应用,耐碳青霉烯类鲍曼不动杆菌(carbapenem resistant Acinetobacter baumannii,CRAB)比例不断上升,甚至出现多重耐药(multiple-drugresistance,MDR)和泛耐药(pan-drug resistance,PDR)菌株,引起全球多处的重症监护病房发生暴发流行.该耐药性的产生是由多种耐药机制共同作用的结果,其中,具有碳青霉烯类水解活性A类酶的出现,使抗感染治疗步入困境,成为该菌耐药机制之一.迄今为止,已在鲍曼不动杆菌中发现多种类型的A类酶,如GES、KPC、SHV、TEM、PER、VEB和CTX-M等,具有水解青霉素类、早期的头孢菌素类、单酰胺类、以及亚胺培南和美罗培南的特性,但可被克拉维酸和三唑巴坦抑制.

Objective To construct mutant strains of Klebsiella pneumoniae with ampG gene dele-tion by homologous recombination and to evaluate the role of ampG gene in inducing the expression of AmpC enzyme.Methods Polymerase chain reaction ( PCR) was used to amplify the upstream and downstream fragments of ampG gene.The gene splicing by overlap extension PCR ( SOE-PCR) technique was used to construct the fusion fragment , which was then ligated into the temperature sensitive suicide vector pKO 3-km after enzyme digestion as pKO3-km-ΔampG.To achieve allelic exchange , the plasmid pKO3-km-ΔampG was introduced into Kp1 and Kp NTUH-K2044 strains by electroporation .The mutant strains of Klebsiella pneu-moniae with ampG gene deletion were screened out .The plasmid pACYC184-ampCR was introduced into the Kp NTUH-K2044 wild-type strain and its mutant strain with ampG gene deletion to make them harbor the gene encoding AmpC enzyme .The disk diffusion method was used to evaluate the effects of ampG gene on the expression of AmpC enzyme in Klebsiella pneumoniae strains with cefoxitin as the inducer .Results The recombinant plasmid pKO3-km-ΔampG was constructed successfully .The mutant strains of Klebsiella pneu-moniae with ampG gene deletion were constructed as verified by PCR and DNA sequencing .Compared with the Kp1 wild type strain, no AmpC enzyme was produced by the ampG gene knock-out Kp1 strain.The Kp NTUH-K2044 strain could produce AmpC enzyme , while the mutant strain of Kp NTUH-K2044 with ampG gene deletion could not after introduced the pACYC 184-ampCR plasmid .Conclusion The mutant strain of Klebsiella pneumoniae with ampG gene deletion was successfully constructed .The Klebsiella pneumonia strain without the ampG gene could not produce the AmpC enzyme .

OBJECTIVE To investigate antibiotic resistant profile of IRAB ,the epidemiology of OXA carbapene‐mase and ISAba1 in clinical isolates ,so as to provide the reference for rational use of antibiotics and control of nosocomial infections .METHODS Totally 140 strains of Acinetobacter baumannii isolated from Oct .2011 to Jun . 2012 in the hospital were collected .A multiplex PCR was designed to investigate the genotype of the ITS sequence of ABA .Antibiotic resistance was tested by K‐B method .Carbapenemase strains were primarily screened by the modified Hodges test .The OXA carbapenemase and integrase gene were determined by multiplex PCR .A single PCR was carried out to detect the ISAba1 upstream of blaOXA‐23‐like and blaOXA‐51‐like .Some positive strains of blaOXA‐23‐like ,blaOXA‐24‐like ,blaOXA‐51‐like were sequenced and gene determined by PCR .RESULTS Of 140 strains identified by multiplex PCR ,137 strains were ABA ,124 strains were IRBA and 13 were ISBA . The resistance rate of IRAB to most antibacterials was over 90 .00% .The gene detection rate of blaOXA‐51‐like , blaOXA‐23‐like were 91 .24% and 94 .89% respectively . No blaOXA‐58‐like gene was found . After whole genome sequencing ,the genotypes were OXA‐23 ,OXA‐72and OXA‐66 ,respectively .CONCLUSION ABA from Harbin region is seriously resistant to all the antibiotics ,and OXA‐23 is the major cause .ISAba1 plays an important role in the expression of OXA‐23 ..

肺炎克雷伯菌是一种能够引起一系列严重疾病的条件致病菌,近年来随着耐药肺炎克雷伯菌的广泛传播,real-time PCR技术在疾病早期检测其耐药基因显得尤为重要.本文就real-time PCR技术在诊断肺炎克雷伯菌耐药性方面的应用进行总结.

Staphylococcus aureus belongs to one of the most common bacteria causing healthcare and community associated infections in China, but their molecular characterization has not been well studied. From May 2011 to June 2012, a total of 322 non-duplicate S. aureus isolates were consecutively collected from seven tertiary care hospitals in seven cities with distinct geographical locations in China, including 171 methicillin sensitive S. aureus (MSSA) and 151 MRSA isolates. All isolates were characterized by spa typing. The presence of virulence genes was tested by PCR. MRSA were further characterized by SCCmec typing. Seventy four and 16 spa types were identified among 168 MSSA and 150 MRSA, respectively. One spa type t030 accounted for 80.1% of all MRSA isolates, which was higher than previously reported, while spa-t037 accounted for only 4.0% of all MRSA isolates. The first six spa types (t309, t189, t034, t377, t078 and t091) accounted for about one third of all MSSA isolates. 121 of 151 MRSA isolates (80.1%) were identified as SCCmec type III. pvl gene was found in 32 MSSA (18.7%) and 5 MRSA (3.3%) isolates, with ST22-MSSA-t309 as the most commonly identified strain. Compared with non-epidemic MRSA clones, epidemic MRSA clones (corresponding to ST239) exhibited a lower susceptibility to rifampin, ciprofloxacin, gentamicin and trimethoprim-sulfamethoxazole, a higher prevalence of sea gene and a lower prevalence of seb, sec, seg, sei and tst genes. The increasing prevalence of multidrug resistant spa-t030 MRSA represents a major public health problem in China.

The present study aimed to investigate the regulatory mechanism of the AmpC enzyme by analyzing the construction and function of AmpCR, AmpE and AmpG genes in the Dhahran (DHA)‑1 plasmid of Klebsiella pneumoniae (K. pneumoniae). The production of AmpC and extended‑spectrum β‑lactamase (ESBL) were determined following the cefoxitin (FOX) inducing test for AmpC, preliminary screening and confirmation tests for ESBL in 10 DHA‑1 plasmid AmpC enzymes of K. pneumoniae strains. AmpCR, AmpD, AmpE and AmpG sequences were analyzed by polymerase chain reaction. The pACYC184‑X plasmid analysis system was established and examined by regulating the pAmpC enzyme expression. The electrophoretic bands of AmpCR, AmpD, AmpE and AmpG were expressed. Numerous mutations in AmpC + AmpR (AmpCR) and in the intergenic region cistron of AmpC‑AmpR, AmpD, AmpE and AmpG were observed. The homology of AmpC and AmpR, in relation to the Morganella morganii strain, was 99%, which was determined by comparing the gene sequences of Kp1 with those of Kp17 AmpCR. The specific combination of AmpR and labeled probe demonstrated a band retarded phenomenon and established a spatial model of AmpR. All the enzyme production strains demonstrated Val93→Ala in AmpG; six transmembrane domains were found in AmpE in all strains, with the exception of Kp1 and Kp4, which had only three transmembrane segments that were caused by mutation. The DHA‑1 plasmid AmpC enzymes encoded by plasmid are similar to the inducible chromosomal AmpC enzymes, which are also regulated by AmpD, AmpE, AmpR and AmpG.

近年来,临床抗生素的使用量不断增加,抗生素不合理应用的现象也日趋严重,由抗生素选择性压力带来的临床耐药菌株不断增多.对头孢菌素类、氨基糖苷类、喹诺酮类等常用抗菌药物多重耐药,甚至对包括亚胺培南或美罗培南在内的碳青霉烯类药物耐药的多重耐药菌不断出现,给临床治疗带来极大困难[1-2],已经引起了临床医师和微生物学工作者对多重耐药菌的广泛关注.

Objective To investigate the drug resistance and molecular epidemiological characteristics of Staphylococcus aureus( SA) isolates from the 2nd Affiliated Hospital of Harbin Medical University,so as to provide basis for control of Staphylococcus aureus epidemic. Methods The susceptibility of 60 SA isolates was detected by disk diffusion method. The presence of mecA gene,pvl gene and some toxin genes in these SA isolates were detected by PCR,and the 60 SA isolates were also analyzed by spa typing. Results In the 60 SA isolates,34 strains were methicillin-resistant Staphylococcus aureus( MRSA,56. 7%) and 26 strains were methicillin-sensitive Staphylococcus aureus( MSSA,43. 3%). Compared with MSSA,MRSA were less sensitive to rifampin,levofloxacin,moxifloxacin,ciprofloxacin,tetracycline and gentamicin( P 0. 05). Toxin gene sea was found in 50 strains,seb was found in 4 strains,eta was found in 1 strain,pvl was found in 1 strain,and all were detected from CA-MSSA,while the toxin genes sec,tst,sasX were not detected. spa typing showed that there were 31 strains of t030,1 strain of t748,and two untyped strains in MRSA,while t078,t571 and t1451 were the first three spa types in MSSA,with the proportion of 23. 1%,7. 7% and 7. 7%,respectively. A new spa type t12444 was detected in 1 MSSA strain. Conclusion The MSSA isolates show high genetic diversity. t030 is the most prevalent spa type among clinical MRSA strains,it is necessary to strengthen monitoring and prevention and control measures to reduce SA infection.

实验诊断学作为五年制临床医学专业必修课,在临床五年制医学生培养中占有很大比重,本课程涉及多学科,是联系内科学、外科学、诊断学等多学科的桥梁.其中的实验课主要是理论教学和实践训练教学,以培养学生的动手能力,使学生感性地理解检验结果,从而客观理性地分析结果、分析疾病、确定诊断、指导治疗、判定预后等.但由于陈旧的实验课教学方法较为枯燥,机械计数细胞,长时间等待实验结果,使学生的学习积极性下降.因此,从2009年开始,我院提高了实验课比重,重新编写了实验诊断学实验手册,合理分配了实验内容,取得了较好的效果,现介绍如下.