BALB/c小鼠多重耐药鲍曼不动杆菌肺部感染模型的建立和评估

本研究旨在构建小鼠多重耐药鲍曼不动杆菌肺部感染模型,评估模型小鼠各项炎性指标的变化特征,为研究多重耐药鲍曼不动杆菌的体内致病性提供参考。首先采用聚合酶链反应(polymerase chain reaction,PCR)检测拟建模用鲍曼不动杆菌的毒力基因分布特征,然后通过感染秀丽隐杆线虫筛选建模菌株。结果显示,A5株携带更多的毒力基因,致病性也更强,被选为建模菌株。给小鼠注射环磷酰胺建立免疫抑制,雾化吸入A5株菌液(高、低密度组),监测小鼠状态、肺组织改变、肺组织和肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中菌落计数、外周血白细胞和中性粒细胞计数、细胞因子等的变化。结果显示,感染A5株的小鼠较对照组状态更差,肺充血水肿,肺组织和BALF中出现鲍曼不动杆菌增殖。与低密度组相比,高密度组小鼠外周血白细胞和中性粒细胞增加更明显(白细胞F=78.630,P=0.000;中性粒细胞:F=4.762,P=0.053)。与对照组相比,感染A5株的小鼠血清白细胞介素6(interleukin 6,IL-6)水平升高并趋于稳定(F=14.382, P=0.001),而...     

多重耐药和广泛耐药鲍曼不动杆菌感染治疗的药物选择

鲍曼不动杆菌是医院内感染的重要病原体,其基于内在性和获得性的耐药机制,导致全球抗感染领域面临巨大挑战。目前,针对多重耐药和广泛耐药鲍曼不动杆菌引起的感染尚无有效治疗方案,本文对其可选用的治疗药物及最新进展进行综述。     

Complete Sequence of pABTJ2, A Plasmid from Acinetobacter baumannii MDR-TJ,Carrying Many Phage-like Elements

Acinetobacter baumannii is an important opportunistic pathogeq in hospital, and tile multidrug-resistant isolates of A. haumannii have been increasingly reported i,a recent years. A num- ber of different mechanisms of resistance have been reported, some of which are associated with plasmid-mediated acquisition of genes. Therefore, studies on plasmids in A. haumannii have been a hot issue lately. We have performed complete genome sequencing of A. haumannii MDR-TJ, which is a multidrug-resistant isolate. Finalizing the remaining large scaffold of the previous assem- bly, we found a new plasmid pABTJ2, which carries many phage-like elements. The plasmid pAB- TJ2 is a circular double-stranded DNA molecule, which is 110,967 bp in length. We annotated 125 CDSs from pABTJ2 using IMG ER and ZCURVE_V, accounting lbr 88.28% of the whole plasmid sequence. Many phage-like elements and a tRNA-coding gene were detected in pABTJ2, which is rarely reported among A. haumannii. The tRNA gene is specific for asparagine codon GTT, which may be a small chromosomal sequence picked up through incorrect excision during plasmid forma- tion. The phage-like elements may have been acquired during the integration process, as the GC content of the region carrying phage-like elements was higher than that of the adjacent regions. The finding of phage-like elements and tRNA-coding gene in pABTJ2 may provide a novel insight into the study of A. haumannii pan-plasmidome.     

In vitro activity of antibiotic combinations against multidrug-resistant strains of Acinetobacter baumannii and the effects of their antibiotic resistance determinants

Various combinations of antibiotics are reported to show synergy in treating nosocomial infections with multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii). Here, we studied hospital-acquired outbreak strains of MDR A. baumannii to evaluate optimal combinations of antibiotics. One hundred and twenty-one strains were grouped into one major and one minor clonal group based on repetitive PCR amplification. Twenty representative strains were tested for antibiotic synergy using Etest(?). Five strains were further analyzed by analytical isoelectric focusing and PCR to identify β-lactamase genes or other antibiotic resistance determinants. Our investigation showed that the outbreak strains of MDR A. baumannii belonged to two dominant clones. A combination of colistin and doxycycline showed the best result, being additive or synergistic against 70% of tested strains. Antibiotic additivity was observed more frequently than synergy. Strains possessing the same clonality did not necessarily demonstrate the same response to antibiotic combinations in vitro. We conclude that the effect of antibiotic combinations on our outbreak strains of MDR A. baumannii seemed strain-specific. The bacterial response to antibiotic combinations is probably a result of complex interactions between multiple concomitant antibiotic resistance determinants in each strain.     

Genome sequence of a dominant, multidrug-resistant Acinetobacter baumannii strain, TCDC-AB0715

Acinetobacter baumannii has emerged as a significant nosocomial pathogen worldwide. The increasing trend of carbapenem and fluoroquinolone resistance in A. baumannii severely limits the usage of therapeutic antimicrobial agents. Here we report the genome sequence of a multidrug-resistant A. baumannii strain, TCDC-AB0715, harboring both bla(OXA-23) and bla(OXA-66).     

Genome sequences of three Acinetobacter baumannii strains assigned to the multilocus sequence typing genotypes ST2, ST25, and ST78

Acinetobacter baumannii is an emerging opportunistic gram-negative pathogen responsible for hospital-acquired infections. A. baumannii epidemics described in Europe and worldwide were caused by a limited number of genotypic clusters of multidrug-resistant strains. Here, we report the availability of draft genome sequences for three multidrug-resistant A. baumannii strains assigned to multilocus sequence typing genotypes ST2, ST25, and ST78 that were more frequently isolated during outbreaks occurred in Greece, Italy, Lebanon, and Turkey.     

产β-内酰胺酶鲍曼不动杆菌基因型研究

目的了解临床科室分离的鲍曼不动杆菌耐药状况并分析多重耐药株产β-内酰胺酶基因型,为有效的临床治疗和医院感染控制提供实验室依据。方法采用纸片扩散（K-B）法测定临床分离的312株鲍曼不动杆菌对13种抗菌药物的敏感性,对其中120株多重耐药株用聚合酶链反应（PCR）扩增β-内酰胺酶基因,并对其扩增产物进行基因测序。结果鲍曼不动杆菌对美洛培南、亚胺培南、头孢哌酮/舒巴坦和替卡西林/克拉维酸的耐药率分别为0.0%、1.0%、30.8%和31.4%,其余抗菌药物耐药率在38.5%-80.1%;120株多重耐药菌株中,β-内酰胺酶基因分布AmpC型基因阳性率为66.7%（80/120）、SHV-12型基因阳性率为14.2%（17/120）,PER-1型基因阳性率为16.7%（20/120）,CTX-M-9型基因阳性率为8.2%（10/120）,TEM-1型基因阳性率为9.2%（11/120）;VER-1,CTX-M-1,CTX-M-2,OXA型均阴性。同时携带2种基因型有16株,携带3种基因型有14株。结论临床分离的鲍曼不动杆菌流行株主要是产AmpC酶,且呈多重耐药。     

鲍曼不动杆菌对碳青霉烯类抗生素的耐药性分析

目的了解鲍曼不动杆菌的耐药情况,并检测耐碳青霉烯类鲍曼不动杆菌的耐药基因,为指导临床合理用药、控制院内感染提供依据。方法利用K-B法检测45株鲍曼不动杆菌临床分离株的耐药情况,通过改良Hodge试验、Carba NP试验和EDTA协同试验对多重耐药鲍曼不动杆菌的碳青霉烯酶进行表型检测,并采用PCR技术检测鲍曼不动杆菌携带OXA-23和NDM-1型耐药基因的情况。结果 45株鲍曼不动杆菌临床分离株中共筛出42株多重耐药菌株;利用改良Hodge试验和Carba NP试验检出36株碳青霉烯酶阳性菌株;采用PCR扩增出OXA-23,未扩增出NDM-1。结论鲍曼不动杆菌耐药情况严重,且耐药基因OXA-23携带率高,治疗时应根据药敏试验结果合理用药。     

Susceptibility, phenotypes of resistance, and extended-spectrum β-lactamases in Acinetobacter baumannii strains

Acinetobacter baumannii plays an increasing role in the pathogenesis of infections in humans. The bacilli are frequently isolated from patients treated in intensive care units. A growing resistance to antibiotics is leading to the emergence of strains that are multidrug-resistant and resistant to all available agents. The objective of this study was to assess susceptibility to antibiotics and to determine the presence and current level of the extended-spectrum β-lactamases (ESBLs) and attempt to isolate the Acinetobacter baumannii strain carrying the blaPER gene. A total of 51 strains of A. baumannii identified by phenotypic features were examined. That the strains belonged to the species was confirmed by the presence of the blaOXA-51-like; gene. A broth microdilution method was used for antibacterial susceptibility testing. The occurrence of ESBLs was determined using phenotypic double-disk synergy tests. The PCR technique was used to confirm the presence of the blaPER-1; gene encoding ESBL. The most active antibiotics were meropenem, cefepime and ampicillin/sulbactam, with susceptibility shown by 76.5%, 60.8% and 56.9% of the strains, respectively. The strains exhibited the highest resistance (> 75%) to piperacillin, tetracycline, ciprofloxacin and cefotaxime. Phenotypic tests revealed ESBL mechanism of resistance in approximately 20% of Acinetobacter baumannii isolates. However, the PCR technique did not confirm the presence of the blaPER-1; gene in any of the Acinetobacter baumannii strains examined in our hospital. Acinetobacter baumannii strains demonstrate considerable resistance to many groups of antibiotics. Our findings indicate the involvement of enzymes belonging to families other than PER β-lactamase in resistance to β-lactams in A. baumannii.     

鲍曼不动杆菌无痕基因敲除及hcp基因相关功能

【背景】鲍曼不动杆菌耐药严重,基因敲除是研究细菌毒力与耐药的重要方式。但现有的大部分细菌基因敲除方法基于抗生素抗性筛选,导致不适用于多重耐药菌株的基因敲除。【目的】旨在建立一种非依赖于抗生素抗性筛选的方法,用于敲除多重耐药鲍曼不动杆菌基因。【方法】运用同源重组和自杀载体pMo130-TelR对亚碲酸钾的抗性,使用两步筛选法,构建鲍曼不动杆菌VI型分泌系统溶血素共调节蛋白(Hemolysin-Coregulated Protein,Hcp)基因敲除突变体,并对缺失株的生长能力、细菌竞争能力以及血清抵抗能力进行测试。【结果】通过构建含同源片段的重组pMo130-TelR载体,成功敲除了鲍曼不动杆菌标准株ATCC 17978中的hcp基因,获得了ATCC 17978 hcp基因缺失突变体。突变体生长能力没有显著改变,但细菌竞争能力显著下降,血清抵抗能力显著升高。【结论】pMo130-TelR可成功用于鲍曼不动杆菌无痕基因敲除,对于研究鲍曼不动杆菌的耐药机制等相关问题具有深远意义。     

重症监护病房呼吸机相关性肺炎中鲍曼不动杆菌耐药性的动态分析

目的探讨重症监护病房呼吸机相关性肺炎(VAP)中鲍曼不动杆菌(Acinetobacter baumannii,Ab)的分布特点和耐药性,为临床防治鲍曼不动杆菌感染提供依据。方法对机械通气>48 h的79例患者的气管抽吸物(ETA)进行细菌定量培养和药敏测定。结果回顾性分析79例VAP患者ETA培养出病原菌214株,其中鲍曼不动杆菌79株(36.9%)。鲍曼不动杆菌对常见抗菌药物的耐药率呈上升趋势,出现较多多重耐药和泛耐药菌株。头孢哌酮/舒巴坦和亚胺培南的耐药率分别是55.6%和68.4%,均呈上升趋势。结论鲍曼不动杆菌是VAP常见病原菌之一,鲍曼不动杆菌的耐药性呈上升趋势,特别是多重耐药和泛耐药菌株,应引起高度重视。     

Genetic and phenotypic analysis of Acinetobacter baumannii insertion derivatives generated with a transposome system

Acinetobacter baumannii is a metabolically versatile pathogen that causes severe infections in compromised patients. However, little is known about the genes and factors involved in its basic physiology and virulence properties. Insertion mutagenesis was used to initiate the identification and characterization of some of these factors and genes in the prototype strain 19606. The utilization of the pLOFKm suicide delivery vector, which harbors a suicide mini-Tn10 derivative, proved to be unsuccessful for this purpose. The EZ::TN Tnp transposome system available from Epicentre was then used in conjunction with electroporation to generate isogenic insertional derivatives of A. baumannii 19606. Replica plating showed that 2% of the colonies that grew after electroporation on agar plates without antibiotics also grew in the presence of 40 micro g of kanamycin per ml. DNA hybridization proved that all of the kanamycin-resistant derivatives contained the EZ::TN insertion element, which was mapped to different genomic locations. Replica plating on Simmons citrate agar and microtiter plate-plastic tube assays identified growth- and biofilm-defective derivatives, respectively. The location of the insertion in several of these derivatives was determined by self-ligation of NdeI- or EcoRI-digested genomic DNA and electroporation of Escherichia coli TransforMax EC100D (pir(+)). Sequence analysis of the recovered plasmids showed that some of the A. baumannii 19606 growth-defective derivatives contain insertions within genes encoding activities required for the generation of energy and cell wall components and for the biosynthesis of amino acids and purines. A gene encoding a protein similar to the GacS sensor kinase was interrupted in four derivatives, while another had an insertion in a gene coding for a hypothetical sensor kinase. A. baumannii 19606 derivatives with defective attachment or biofilm phenotypes had insertions within genes that appear to be part of a chaperone-usher transport system described for other bacteria. DNA hybridization experiments showed that the presence of strain 19606 genes encoding regulatory and attachment or biofilm functions is widespread among other A. baumannii clinical isolates.     

Genome sequences of four divergent multidrug-resistant Acinetobacter baumannii strains isolated from patients with sepsis or osteomyelitis

Acinetobacter baumannii is a Gram-negative bacterium that causes nosocomial infections worldwide, with recent prevalence and higher frequency in wounded military personnel. Four A. baumannii strains from the Walter Reed Army Medical Center (WRAMC) isolated between 2008 and 2009 were sequenced, representing diverse, multidrug-resistant isolates from osteomyelitis or septic patients.     

Comparative analysis of Acinetobacters: three genomes for three lifestyles

Acinetobacter baumannii is the source of numerous nosocomial infections in humans and therefore deserves close attention as multidrug or even pandrug resistant strains are increasingly being identified worldwide. Here we report the comparison of two newly sequenced genomes of A. baumannii. The human isolate A. baumannii AYE is multidrug resistant whereas strain SDF, which was isolated from body lice, is antibiotic susceptible. As reference for comparison in this analysis, the genome of the soil-living bacterium A. baylyi strain ADP1 was used. The most interesting dissimilarities we observed were that i) whereas strain AYE and A. baylyi genomes harbored very few Insertion Sequence elements which could promote expression of downstream genes, strain SDF sequence contains several hundred of them that have played a crucial role in its genome reduction (gene disruptions and simple DNA loss); ii) strain SDF has low catabolic capacities compared to strain AYE. Interestingly, the latter has even higher catabolic capacities than A. baylyi which has already been reported as a very nutritionally versatile organism. This metabolic performance could explain the persistence of A. baumannii nosocomial strains in environments where nutrients are scarce; iii) several processes known to play a key role during host infection (biofilm formation, iron uptake, quorum sensing, virulence factors) were either different or absent, the best example of which is iron uptake. Indeed, strain AYE and A. baylyi use siderophore-based systems to scavenge iron from the environment whereas strain SDF uses an alternate system similar to the Haem Acquisition System (HAS). Taken together, all these observations suggest that the genome contents of the 3 Acinetobacters compared are partly shaped by life in distinct ecological niches: human (and more largely hospital environment), louse, soil.     

Identification of Streptomyces sp. KH29, which produces an antibiotic substance processing an inhibitory activity against multidrug-resistant Acinetobacter baumannii

The Actinomycete strain KH29 is antagonistic to the multidrug-resistant Acinetobacter baumannii. Based on the diaminopimelic acid (DAP) type, and the morphological and physiological characteristics observed through the use of scanning electron microscopy (SEM), KH29 was confirmed as belonging to the genus Streptomyces. By way of its noted 16S rDNA nucleotide sequences, KH29 was found to have a relationship with Streptomyces cinnamonensis. The production of an antibiotic from this strain was found to be most favorable when cultured with glucose, polypeptone, and yeast extract (PY) medium for 6 days at 27 degrees C. The antibiotic produced was identified, through comparisons with reported spectral data including MS and NMR as a cyclo(L-tryptophanyl-L-tryptophanyl). Cyclo(L-Trp-L-Trp), from the PY cultures of KH29, was seen to be highly effective against 41 of 49 multidrugresistant Acinetobacter baumannii. Furthermore, cyclo(LTrp- L-Trp) had antimicrobial activity against Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Saccharomyces cerevisiae, Aspergillus niger, and Candida albicans, However, it was ineffective against Streptomyces murinus.     

Draft genome sequence of a multidrug-resistant Acinetobacter baumannii ST15 (CC15) isolated from Brazil

Acinetobacter baumannii is an important pathogen frequently associated with nosocomial outbreaks around the world. In Brazil, A. baumannii has become particularly problematic because of its prevalence and the carbapenems resistance. Here, we report the draft genome sequence of a multidrug-resistant A. baumannii (ST15/CC15) isolated in 2009 from the state of Espírito Santo (Southeast Brazil). We observed important resistance determinant genes in an estimated genome size of 4,102,788 bp with 3,862 predicted coding regions. A detailed report of the genomic data analysis might help to understand the specific features of highly successful strains belonged to a relevant complex clonal in different Brazilian geographical regions.     

外排泵介导鲍曼不动杆菌耐药性的研究进展

目的鲍曼不动杆菌的多重耐药性问题日趋严重,该菌外膜上外排泵过表达是导致其耐药性的重要机制。详尽地研究多药外排泵的机制以及寻找阻断其功能的外排泵抑制剂,将为多耐药鲍曼不动杆菌的治疗开辟新的路径。本文就近年来鲍曼不动杆菌外排泵的研究现状进行综述,着重描述多药外排泵RND家族的耐药谱特征及其表达调控机制,同时,还阐述了MFS和MATE家族外排泵的研究进展。     

Differences in Acinetobacter baumannii strains and host innate immune response determine morbidity and mortality in experimental pneumonia

Despite many reports documenting its epidemicity, little is known on the interaction of Acinetobacter baumannii with its host. To deepen our insight into this relationship, we studied persistence of and host response to different A. baumannii strains including representatives of the European (EU) clones I-III in a mouse pneumonia model. Neutropenic mice were inoculated intratracheally with five A. baumannii strains and an A. junii strain and at several days morbidity, mortality, bacterial counts, airway inflammation, and chemo- and cytokine production in lungs and blood were determined. A. baumannii RUH875 and RUH134 (EU clone I and II, respectively) and sporadic strain LUH8326 resulted in high morbidity/mortality, whereas A. baumannii LUH5875 (EU clone III, which is less widespread than clone I and II) caused less symptoms. A. baumannii type strain RUH3023(T) and A. junii LUH5851 did not cause disease. All strains, except A. baumannii RUH3023(T) and A. junii LUH5851, survived and multiplied in the lungs for several days. Morbidity and mortality were associated with the severity of lung pathology and a specific immune response characterized by low levels of anti-inflammatory (IL-10) and specific pro-inflammatory (IL-12p40 and IL-23) cytokines at the first day of infection. Altogether, a striking difference in behaviour among the A. baumannii strains was observed with the clone I and II strains being most virulent, whereas the A. baumannii type strain, which is frequently used in virulence studies appeared harmless.     

多药耐药鲍曼不动杆菌老年患者分离株发现ADC型β-内酰胺酶基因新的变异型

目的调查多药耐药鲍曼不动杆菌老年患者分离株(MDR-ABA-5077)β-内酰胺酶基因分布情况。方法 MDR-ABA-5077株分离自宁波市医疗中心李惠利医院2009年7月住院老年患者,采用聚合酶联反应(PCR)及序列分析的方法分析21种β-内酰胺酶基因。结果本株MDR-ABA共检出3种β-内酰胺酶基因:TEM、SHV、ADC,其余18种β-内酰胺酶基因未检出。ADC阳性基因测得序列经BLAST比对与已在GenBank登录的ADC型AmpC均不相同,经与GenBank登录的ADC型序列分子进化分析,确认为ADC型β-内酰胺酶新的变异型。结论本株MDR-ABA多种β-内酰胺类药物耐药与产TEM、SHV、ADC等3种β-内酰胺酶相关。     

Comparative proteomics of inner membrane fraction from carbapenem-resistant Acinetobacter baumannii with a reference strain

Acinetobacter baumannii has been identified by the Infectious Diseases Society of America as one of the six pathogens that cause majority of hospital infections. Increased resistance of A.baumannii even to the latest generation of β-lactams like carbapenem is an immediate threat to mankind. As inner-membrane fraction plays a significant role in survival of A.baumannii, we investigated the inner-membrane fraction proteome of carbapenem-resistant strain of A.baumannii using Differential In-Gel Electrophoresis (DIGE) followed by DeCyder, Progenesis and LC-MS/MS analysis. We identified 19 over-expressed and 4 down-regulated proteins (fold change>2, p<0.05) in resistant strain as compared to reference strain. Some of the upregulated proteins in resistant strain and their association with carbapenem resistance in A.baumannii are: i) β-lactamases, AmpC and OXA-51: cleave and inactivate carbapenem ii) metabolic enzymes, ATP synthase, malate dehydrogenase and 2-oxoglutarate dehydrogenase: help in increased energy production for the survival and iii) elongation factor Tu and ribosomal proteins: help in the overall protein production. Further, entry of carbapenem perhaps is limited by controlled production of OmpW and low levels of surface antigen help to evade host defence mechanism in developing resistance in A.baumannii. Present results support a model for the importance of proteins of inner-membrane fraction and their synergistic effect in the mediation of resistance of A.baumannii to carbapenem.