鲍曼不动杆菌临床菌株生物膜形成能力的研究

目的对鲍曼不动杆菌临床菌株的生物膜形成能力进行对比研究,并分析生物膜形成的一些可能的影响因素。方法利用在聚苯乙烯板上构建生物膜的技术对51株全耐药的鲍曼不动杆菌的生物膜形成能力进行检测,同时对分离自下呼吸道和无菌体液的各20株鲍曼不动杆菌的生物膜形成能力进行研究,然后对新近报道的鲍曼不动杆菌生物膜形成相关基因abaI在所有菌株中的分布情况进行检测。结果 51株全耐药的鲍曼不动杆菌中35株(68.6%)可以形成生物膜,并且形成生物膜的能力较强。50%(10/20)分离自下呼吸道的鲍曼不动杆菌能够形成生物膜,20株无菌体液中分离的菌株仅有1株可以形成生物膜。78.0%(71/91)鲍曼不动杆菌中abaI基因扩增阳性。结论分离自下呼吸道的鲍曼不动杆菌临床菌株有较强的生物膜形成能力,abaI基因广泛存在于鲍曼不动杆菌临床菌株中。临床在治疗鲍曼不动杆菌感染的同时需要考虑其在感染部位形成生物膜的因素,可能在治疗的同时有必要加入一些对生物膜有穿透性的药物。     

Effect of a Novel Podophage AB7-IBB2 on Acinetobacter baumannii Biofilm

Biofilm formation in Acinetobacter baumannii is a common cause of nosocomial infections in humans. Clinical devices and abiotic surfaces are important sites of colonization leading to formation of biofilms. Such infections are often resistant to multiple antibiotic therapies, and hence there is need for an effective mode of control. Herein, we describe the isolation, characterization of a new lytic bacteriophage of A.?baumannii and its effect on biofilm. The phage AB7-IBB2, with a genome size of about 170?kb was identified to be of family Podoviridae as revealed by transmission electron microscopy. It had an isometric head (35?nm) and a short tail (7?nm). It lysed 19/39 (49?%) clinical isolates of A.?baumannii. Rapid adsorption (>99?% adsorbed in 4?min), a latency period of 25?min and a burst size 22?PFU/infected cell was observed. The phage could inhibit A.?baumannii biofilm formation and disrupt preformed biofilm as well. The phage has promising potential to be considered as a candidate biocontrol agent for A.?baumannii infections.     

Adherence and motility characteristics of clinical Acinetobacter baumannii isolates

Acinetobacter baumannii continues to be a major health problem especially in hospital settings. Herein, features that may play a role in persistence and disease potential were investigated in a collection of clinical A. baumannii strains from Australia. Twitching motility was found to be a common trait in A. baumannii international clone I strains and in abundant biofilm formers, whereas swarming motility was only observed in isolates not classified within the international clone lineages. Bioinformatic analysis of the type IV fimbriae revealed a correlation between PilA sequence homology and motility. A high level of variability in adherence to both abiotic surfaces and epithelial cells was found. We report for the first time the motility characteristics of a large number of A. baumannii isolates and present a direct comparison of A. baumannii binding to nasopharyngeal and lung epithelial cells.     

Acinetobacter baumannii biofilms: Variations among strains and correlations with other cell properties

Acinetobacter baumannii is an opportunistic pathogen that causes serious infections in humans by colonizing and persisting on surfaces normally found in hospital settings. The capacity of this pathogen to persist in these settings could be due to its ability to form biofilms on inanimate surfaces. This report shows that although the ATCC 19606(T) type strain and 8 different clinical isolates form biofilms, there are significant variations in the cell density and microscopic structures of these cell aggregates, with 3 of the isolates forming pellicles floating on the surface of stagnant broth cultures. PCR indicated that, like ATCC 19606(T), all 8 clinical isolates harbor all the genetic components of the CsuA/BABCDE chaperone-usher pili assembly system, which is needed for biofilm formation on plastic. Pili detection in cells of all strains examined supports the presence and function of a pilus assembly system. However, only one of them produced the putative ATCC 19606(T) CsuA/B pilin subunit protein. Hydrophobicity tests and motility assays also showed significant variations among all tested strains and did not result in direct correlations between the biofilm phenotype and cell properties that could affect biofilm formation on abiotic surfaces. This lack of correlation among these 3 phenotypes may reflect some of the variations already reported with this pathogen, which may pose a challenge in the treatment of the infections this pathogen causes in humans using biofilm formation on abiotic surfaces as a target.     

N-acyl homoserine lactone mediated interspecies interactions between A. baumannii and P. aeruginosa

Acinetobacter baumannii and Pseudomonas aeruginosa are pathogens capable of colonizing the same infection sites and employing N-acyl homoserine lactone (AHL) based quorum-sensing systems to co-ordinate biofilm formation. Hence, the effect of P. aeruginosa AHLs on biofilm formation by A. baumannii and vice versa were investigated using the biofilm impaired quorum sensing mutants, A. baumannii M2 (abaI::Km) and P. aeruginosa PAO-JP2. Complementing the mutants with heterologous, extracted and pure AHLs increased biofilm mass significantly. The surface area coverage and biovolume also increased significantly as observed by confocal scanning laser microscopy which corroborated scanning electron microscope analysis. Autoinducer synthase gene promoters of A. baumannii, P( abaI)-lacZ, and P. aeruginosa, P( lasI)-lacZ, were induced (p?相似文献   

Cell surface properties of two differently virulent strains of Acinetobacter baumannii isolated from a patient

The aim of this study was to unravel, by focusing on cell surface properties, the underlying virulence factors contributing to the difference in the pathogenicity observed in two Acinetobacter baumannii strains isolated from the same patient. The two strains were phenotypically different: (i) a mucoid strain (AB-M), highly virulent in a mouse model of pneumonia, and (ii) a nonmucoid strain (AB-NM), moderately virulent in the same model. The study of the cell surface properties included the microbial adhesion to solvents method, the measurement of the electrophoretic mobility of bacteria, the analysis of biofilm formation by calcofluor white staining, the adherence to silicone catheters, and scanning electron microscopy. The AB-NM strain was more hydrophobic, more adherent to silicone catheters, and produced more biofilm than the AB-M strain. Scanning electron microscopy showed bacterial cells with a rough surface and the formation of large cell clusters for AB-NM whereas the AB-M strain had a smooth surface and formed only a few cell clusters. Contrary to the results of most previous studies, cell surface properties were not correlated to the virulence described in our experimental model, indicating that mechanisms other than adherence may be involved in the expression of A.?baumannii virulence.     

Extracellular stress and lipopolysaccharide modulate Acinetobacter baumannii surface-associated motility

Acinetobacter baumannii is a nosocomial bacterial pathogen, and infections attributed to this species are further complicated by a remarkable ability to acquire antimicrobial resistance genes and to survive in a desiccated state. While the antibiotic resistance and biofilm formation of A. baumannii is well-documented, less is known about the virulence attributes of this organism. Recent studies reported A. baumannii strains display a motility phenotype, which appears to be partially dependent upon Type IV pili, autoinducer molecules, and the response to blue light. In this study, we wanted to determine the prevalence of this trait in genetically diverse clinical isolates, and any additional required factors, and environmental cues that regulate motility. When strains are subjected to a wide array of stress conditions, A. baumannii motility is significantly reduced. In contrast, when extracellular iron is provided or salinity is reduced, motility is significantly enhanced. We further investigated whether the genes required for the production of lipopolysaccharide (lpsB) and K1 capsule (epsA/ptk) are required for motility as demonstrated in other Gram-negative bacteria. Transposon mutagenesis resulted in reduced motility by the insertion derivatives of each of these genes. The presence of the parental allele provided in trans, in the insertion mutant background, could only restore motility in the lpsB mutant. The production of core LPS directly contributes to the motility phenotype, while capsular polysaccharide may have an indirect effect. Further, the data suggest motility is regulated by extracellular conditions, indicating that A. baumannii is actively sensing the environment and responding accordingly.     

金属离子铁和锌对鲍曼不动杆菌的抗菌作用机制研究进展

由于抗菌药物的开发周期越来越长,远远赶不上细菌耐药的发展速度,临床鲍曼不动杆菌多重耐药与泛耐药现象日益严重。因此,人们越来越关注对抗菌药物以外的抗菌物质的开发,尤其是从生存条件方面来研究抑制耐药菌活性的方法,如金、银、铜等金属离子对鲍曼不动杆菌的作用。本文主要综述铁、锌等金属离子及其螯合物对鲍曼不动杆菌的抗菌作用。铁、锌等金属离子通过与一系列酶的协同作用,调控外排泵或影响生物膜形成及其黏附性等来抑制细菌生长。此外,一些非必需金属如金、银、钯等对鲍曼不动杆菌也具有很强的毒性,有良好的抗菌和降低耐药率的效果,可作为医疗留置器械的抗菌涂层等来预防感染。     

Complete genome sequence of multidrug-resistant Acinetobacter baumannii strain 1656-2, which forms sturdy biofilm

Acinetobacter baumannii is a Gram-negative bacterium causing nosocomial infections worldwide. To gain quick insight into the molecular basis of biofilm formation in A. baumannii, we determined the complete genome sequence of A. baumannii strain 1656-2, which forms sturdy biofilm and is resistant to multiple drugs.     

Study of the formation of a biofilm by clinical strains of Staphylococcus aureus

A study on biofilm formation was carried out using five methicillin-sensitive [MSSA] and five methicillin-resistant [MRSA] strains of S. aureus. In each group, there were four strains isolated from patients from Kinshasa (Democratic Republic of Congo, DRC) and one reference strain. All of the strains were hydrophobic. The adherence of the bacteria to an abiotic surface was studied with the Biofilm Ring Test (BFRT?) and the crystal violet staining method (CVSM). Both techniques showed that eight of the strains formed biofilms within 2-3 h. The extent of the biofilm formed by one strain could only be observed with the CVSM. Periodate prevented the formation of biofilms and, in separate experiments, destroyed the biofilm pre-formed by the MSSA reference, but not those pre-formed by the clinical strains. Proteinase K destroyed all pre-formed biofilms. Six of the strains were icaA+; the clinical MSSA strains were not. The results also indicated different mechanisms of biofilm development between MSSA and MRSA clinical strains. The BFRT? and the CVSM are complementary techniques to study the adhesion of bacteria and the development of biofilms.     

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.     

乳酸锌和氟化亚锡对铜绿假单胞菌、鲍曼不动杆菌和变异链球菌生物被膜的抑制作用

乳酸锌(Zn lactate·3H_2O)和氟化亚锡(SnF_2)常作为牙膏中的活性物质添加剂用来预防龋齿及口腔生物被膜的形成。文中评估了Zn lactate·3H_2O和SnF_2对铜绿假单胞菌、鲍曼不动杆菌和变异链球菌生物被膜的作用。对铜绿假单胞菌PAO1生物被膜的抑制实验证实乳酸锌和氟化亚锡都具有抑制其生物被膜的功能,联用效果尤佳。乳酸锌通过干扰胞外多糖基质网的形成起作用,而氟化亚锡则可以明显降低生物被膜的生物量。更为重要的是,工作浓度的两种化合物联用几乎可以完全抑制3种实验菌株生物被膜的形成。     

Inhibitory Effect on In Vitro Streptococcus oralis Biofilm of a Soda-Lime Glass Containing Silver Nanoparticles Coating on Titanium Alloy

This paper reports the effect of soda-lime-glass-nAg coating on the viability of an in vitro biofilm of Streptococcus oralis. Three strains (ATCC 35037 and two clinical isolates from periodontitis patients) were grown on coated with glass, glass containing silver nanoparticles, and uncoated titanium alloy disks. Two different methods were used to quantify biofilm formation abilities: crystal violet staining and determination of viable counts. The influence of the surface morphology on the cell attachment was studied. The surface morphology was characterized by scanning electron microscopy (SEM) and using a profilometer. SEM was also used to study the formation and the development of biofilm on the coated and uncoated disks. At least a >99.7% inocula reduction of biofilm respect to titanium disks and also to glass coated disks was observed in the glass-nAg coated disks for all the studied strains. A quantitative evaluation of the release of silver was conducted in vitro to test whether and to what extend the biocidal agent (silver) could leach from the coating. These findings suggest that the biofilm formation of S. oralis strains is highly inhibited by the glass-nAg and may be useful for materials which require durable antibacterial effect on their surfaces, as it is the case of dental implants.     

Proteomic and functional analyses reveal a unique lifestyle for Acinetobacter baumannii biofilms and a key role for histidine metabolism

Biofilm formation is one of the main causes for the persistence of Acinetobacter baumannii, a pathogen associated with severe infections and outbreaks in hospitals. Here, we performed comparative proteomic analyses (2D-DIGE and MALDI-TOF/TOF and iTRAQ/SCX-LC-MS/MS) of cells at three different conditions: exponential, late stationary phase, and biofilms. These results were compared with alterations in the proteome resulting from exposure to a biofilm inhibitory compound (salicylate). Using this multiple-approach strategy, proteomic patterns showed a unique lifestyle for A. baumannii biofilms and novel associated proteins. Several cell surface proteins (such as CarO, OmpA, OprD-like, DcaP-like, PstS, LysM, and Omp33), as well as those involved in histidine metabolism (like Urocanase), were found to be implicated in biofilm formation, this being confirmed by gene disruption. Although l-His uptake triggered biofilms efficiently in wild-type A. baumannii, no effect was observed in Urocanase and OmpA mutants, while a slight increase was observed in a CarO deficient strain. We conclude that Urocanase plays a crucial role in histidine metabolism leading to biofilm formation and that OmpA and CarO can act as channels for L-His uptake. Finally, we propose a model in which novel proteins are suggested for the first time as targets for preventing the formation of A. baumannii biofilms.     

Study of the formation of a biofilm by clinical strains of Staphylococcus aureus

A study on biofilm formation was carried out using five methicillin-sensitive [MSSA] and five methicillin-resistant [MRSA] strains of S. aureus. In each group, there were four strains isolated from patients from Kinshasa (Democratic Republic of Congo, DRC) and one reference strain. All of the strains were hydrophobic. The adherence of the bacteria to an abiotic surface was studied with the Biofilm Ring Test (BFRT®) and the crystal violet staining method (CVSM). Both techniques showed that eight of the strains formed biofilms within 2–3 h. The extent of the biofilm formed by one strain could only be observed with the CVSM. Periodate prevented the formation of biofilms and, in separate experiments, destroyed the biofilm pre-formed by the MSSA reference, but not those pre-formed by the clinical strains. Proteinase K destroyed all pre-formed biofilms. Six of the strains were icaA⁺; the clinical MSSA strains were not. The results also indicated different mechanisms of biofilm development between MSSA and MRSA clinical strains. The BFRT® and the CVSM are complementary techniques to study the adhesion of bacteria and the development of biofilms.     

Identification of virulence markers in clinically relevant strains of Acinetobacter genospecies

Nine Acinetobacter strains from patients and hospital environment were analyzed for virulence markers, quorum sensing signal production, and the presence of luxI and luxR genes. The strains had several properties in common: growth in iron limited condition, biofilm formation, and no active protease secretion. Significantly higher catechol production was determined in patient isolates (P < 0.03), but other invasiveness markers, such as lipase secretion, amount of biofilm, cell motility, antibiotic resistance, and hemolysin production, showed large variability. Notably, all members of the so-called A. calcoaceticus-A. baumannii complex, regardless of whether the source was a patient or environmental, secreted mediumto long-chain N-acyl homoserine lactones (AHL) and showed blue light inhibition of cell motility. In these strains, a luxI homologue with a homoserine lactone synthase domain and a luxR putative regulator displaying the typical AHL binding domain were identified.     

一株工业腐败物中魏氏柠檬酸杆菌的分离鉴定及产生物膜特性分析

【目的】分离和鉴定工业腐败物中高产细菌生物膜菌株,并明确该菌的部分产膜特性。【方法】通过微孔板结晶紫染色法对分离的菌株进行产膜能力评价,根据菌落形态、生理生化特性和16S rRNA序列的系统进化树分析进行菌株鉴定;同时利用扫描电子显微镜(SEM)和结晶紫染色法分别研究材料及温度对该菌产膜特性和能力的影响。【结果】筛选出一株高产细菌生物膜菌株,经鉴定该菌为魏氏柠檬酸杆菌;其在玻璃、不锈钢和聚氯乙烯(PVC)材料表面均能形成生物膜;温度条件显著影响产膜能力,在30°C时,菌株在PVC材料表面形成生物膜能力最强。【结论】工业腐败物中含有高产细菌生物膜菌株,并且产膜受附着物和温度影响。     

两种临床标本中耐甲氧西林的金黄色葡萄球菌生物膜形成能力的检测与其耐药特征的研究

目的研究两种临床标本来源中耐甲氧西林的金黄色葡萄球菌（MRSA）生物膜形成能力和抗菌药物耐药率,并探讨二者的关系,为临床检测和治疗提供依据。方法收集温州医学院附属第二医院2009年1月至2010年12月的分离鉴定的MRSA共128株,其中脓液59株,痰液69株;采用刚果红平板检测多糖粘附素（PIA）,半定量粘附试验检测生物膜表型,PCR检测icaA基因结果用z。分割法进行统计分析。结果128株MRSA中生物膜阳性菌株为61株,其中脓液36株、痰液25株,脓液中检出生物膜的阳性率明显高于痰液中检出生物膜的阳性率（x2=7．382,P=0．005）。MRSA对所有B．内酰胺类抗生素全耐药,对万古霉素和利奈唑胺全敏感,痰液中MRSA对利福平、庆大霉素、环丙沙星的耐药率显著高于脓液中MRSA的耐药率（P〈0．05）,但是痰液中MRSA对克林霉素的耐药率要显著低于脓液中MRSA的耐药率（P〈0．05）。脓液中生物膜阳性菌株和生物膜阴性菌株对抗菌药物的耐药率差异无统计学意义（P〉0．05）,但是痰液中生物膜阳性菌株对克林霉素的耐药率高于生物膜阴性菌株的耐药率（P〈0．05）,对利福平的耐药率显著低于生物膜阴性菌株的耐药率（P〈0．05）。结论脓液中MRSA的生物膜检出率比痰液高,但是脓液中MRSA的耐药率比痰液中的耐药率要低,MRSA对抗菌药物的耐药谱和生物膜的形成并无直接的关系。