Global comparison of the membrane subproteomes between a multidrug-resistant Acinetobacter baumannii strain and a reference strain

Acinetobacter baumannii causes severe infections in compromised patients. We combined SDS-PAGE, two-dimensional gel electrophoresis and mass spectrometry (LC-MS/MS and MALDI-TOF) to separate and characterize the proteins of the cell envelope of this bacterium. In total, 135 proteins (inner and outer membrane proteins) were identified. In this analysis, we described the expression by this bacterium of RND-type efflux systems and some potential virulence factors. We then compared the membrane subproteome of a clinical multidrug-resistant (MDR) isolate with that of a reference strain. We found that the MDR strain expressed lower levels of the penicillin-binding-protein 1b, produced a CarO protein having different primary and quaternary structures to that of the reference strain, and expressed OmpW isoforms. We also showed that the clinical strain has a high ability to form biofilms consistent with the accumulation of some outer membrane proteins (OMPs) such as NlpE or CsuD that have already been described as involved in bacterial adhesion. These features may partly explain the MDR emergence of the clinical isolate.     

ICU耐碳青霉烯类鲍曼不动杆菌同源性研究

目的探讨重症监护病房（ICU）耐碳青霉烯酶鲍曼不动杆菌（CRAB）之间的同源性,并了解是否存在耐药菌株的克隆流行。方法收集宁波大学附属医院ICU 2010年2月至2011年5月分离到的CRAB 40株,常规药敏试验采用K-B法。用脉冲场凝胶电泳（PFGE）分析其耐药株的同源性。结果阿米卡星的敏感率最高为100%,其次米诺环素敏感率为50%;PFGE结果显示,40株CRAB菌株分为A、B、C三型,A型22株,主要分离时段为2010年2月～3月（16株）和2011年1月-2011年2月（6株）;B型16株,主要分离时段为2010年5月-6月（16株）,C型为C1亚型和C2亚型各1株为散发型。结论调查期间CRAB主要的PFGE基因型为A型和B型菌株克隆流行,流行相关的克隆株可在病区长期生存,从而引起病区持续感染,需要积极采取感染控制措施;同时不同时段主要流行株由克隆株A变为克隆株B,推测同一个病区的流行基因型可能存在流行变迁。     

耐碳青霉烯类鲍曼不动杆菌产OXA酶的研究进展

鲍曼不动杆菌对碳青霉烯类抗生素耐药率逐年增高,其主要耐药机制包括产生水解药物的碳青霉烯酶,外膜孔蛋白的改变,主动外排系统的过度表达和青霉素结合蛋白改变等.其中由于苯唑西林酶(Oxaclillinase,OXA酶)克隆传播导致鲍曼不动杆菌对碳青霉烯类抗生素耐药的爆发流行的报道,已引起大家的广泛关注.本研究对耐碳青霉烯类鲍曼不动杆菌产OXA酶的研究现状作一阐述.     

Dynamics of the Acinetobacter baumannii inner membrane under exogenous polyunsaturated fatty acid stress

Exogenous polyunsaturated fatty acids (PUFAs) are readily incorporated into the synthesis pathways of A. baumannii membrane phospholipids, where they contribute to reduced bacterial fitness and increased antimicrobial susceptibility. Here we examine the impact of PUFA membrane modification on membrane organisation and biophysical properties using coarse grained MARTINI simulations of chemically representative membrane models developed from mass-spectrometry datasets of an untreated, arachidonic acid (AA) treated and docosahexaenoic acid (DHA) treated A. baumannii membranes. Enzymatic integration of AA or DHA into phospholipids of the A. baumannii membrane resulted in modulation of membrane biophysical properties. Membrane thickness decreased slightly following PUFA treatment, concomitant with changes in the lateral area per lipid of each lipid headgroup class. PUFA treatment resulted in a decrease in membrane ordering and an increase in lipid lateral diffusion. Changes in lateral membrane organisation were observed in the PUFA treated membranes, with a concurrent increase in ordered cardiolipin domains and disordered PUFA-containing domains. Notably, separation between ordered and disordered domains was enhanced and was more pronounced for DHA relative to AA, providing a possible mechanism for greater antimicrobial action of DHA relative to AA observed experimentally. Furthermore, the membrane active antimicrobial, pentamidine, preferentially adsorbs to cardiolipin domains of the A. baumannii model membranes. This interaction, and membrane penetration of pentamidine, was enhanced following PUFA treatment. Cumulatively, this work explores the wide-ranging effects of PUFA incorporation on the A. baumannii membrane and provides a molecular basis for bacterial inner membrane disruption by PUFAs.     

呼吸科住院患者耐碳青霉烯鲍曼不动杆菌抗菌药物敏感性及其分子特征

目的调查深圳市人民医院呼吸科住院患者耐碳青霉烯鲍曼不动杆菌（CRAB）的抗菌药物敏感性和耐药分子机制,以及克隆流行情况。方法收集2010年深圳市人民医院呼吸科住院患者临床分离CRAB29株,琼脂稀释法测定亚胺培南等15种抗菌药对CRAB的最低抑菌浓度（MIC）,PCR和DNA测序分析CRAB碳青霉烯酶基因型,脉冲场凝胶电泳（PFGE）分析菌株同源性。结果多粘菌素B对29株CRAB抗菌活性最强,敏感性100％,MIC50／MIC90为1／1μg/mL,其次米诺环素,敏感性96．6％,MIC50／MIC90为4／4μg/mL,替加环素中介率高达96．6％,MIC50／MIC90为4／4μg/mL。96．6％（28／29）CRAB携带ISAba1—blaOXA-23-like,对亚胺培南和美罗培南高度耐药,亚胺培南和美罗培南的MIC集中分布在32—64μg／mL;1株携带ISAba1—blaOXA-51-like CRAB,对亚胺培南和美罗培南中度耐药,亚胺培南和美罗培南的MIC分别为4μg/mL和8μg/mL。29株CRAB未发现blaOXA-24-like、blaOXA-143-like、金属酶基因及KPC酶基因。29株CRAB经PFGE分型共分2型,以A型28株（96．6％）为主要流行克隆,均携带ISAba1-blaOXA-23-like。结论2010年我院呼吸科临床分离CRAB主要携带ISAba1—blaOXA-23-like基因,并以克隆播散流行。     

福州地区碳青霉烯类耐药鲍曼不动杆菌碳青霉烯酶基因型研究

目的探讨福州地区碳青霉烯类耐药鲍曼不动杆菌(CRAB)碳青霉烯酶基因型的流行情况。方法收集多家医院临床标本中分离得到的107株CRAB。应用K-B纸片扩散法进行药物敏感试验。采用PCR法检测7种碳青霉烯酶基因,包括OXA-23、OXA-24、OXA-51、OXA-58、IMP-1、IMP-4和VIM-2。结果 107株CRAB对除多粘菌素B、米诺环素外的其他所有常见的抗生素均为耐药。碳青霉烯酶基因OXA-51、OXA-23的检出率分别为100.0%(107/107)和87.9%(94/107)。其他OXA-24、OXA-58、IMP-1、IMP-4和VIM-2基因均未检出。结论福州地区临床分离的CRAB耐药现象严重;表达OXA-23基因是CRAB对碳青霉烯类药物耐药的重要机制之一。     

耐碳青霉烯类鲍曼不动杆菌OXA基因检测及同源性分析

目的 了解台州地区碳青霉烯类耐药鲍曼不动杆菌的耐药性、碳青霉烯酶基因型以及同源性.方法 63株碳青霉烯类耐药鲍曼不动杆菌经VITEK2 Compact进行细菌鉴定及药敏分析,用K-B法复核药敏结果,采用多重PCR扩增分析碳青霉烯酶的基因型;采用脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)分析其同源性.结果 63株菌株为多重耐药菌株,除多粘菌素、阿米卡星、头孢哌酮/舒巴坦外,对其他常用抗生素的耐药率均在70％以上.63株菌株检测出OXA-51基因60株(95.2％),OXA-23基因58株(92.1％),两个基因同时存在的有58株(92.1％).PFGE结果显示碳青霉烯类鲍曼不动杆菌主要分为5个克隆型,其中A、B两个为主型.结论 产OXA酶是台州地区耐碳青霉烯类鲍曼不动杆菌的主要耐药机制之一,其中OXA-23是主要的基因型.     

Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii isolate

Two-component systems (TCSs), typically consisting of a histidine kinase (HK) and a cognate response regulator (RR), are the most common signaling systems in bacteria. Besides paired genes encoding TCSs, there also exists unpaired HKs and orphan RRs. In Streptomyces coelicolor , 13 orphan RRs have been annotated. Because of lack of cognate HKs, little is known as yet about the regulation of orphan RRs. Bioinformatic analysis revealed that several orphan RRs had high amino acid sequence identities with RRs from typical TCSs in S. coelicolor . Among them, the orphan RR SCO3818 and RR SCO0204, which paired with HK SCO0203, showed the highest identity (65%), suggesting that the two RRs might both be under the regulation of SCO0203. Following studies showed that SCO0203 could phosphorylate not only SCO0204 but also SCO3818. Deletion of either sco0203 or sco3818 led to enhanced production of blue-pigmented antibiotic actinorhodin, which indicated a functional correlation between SCO0203 and SCO3818. These results suggested that SCO3818 might be regulated by SCO0203. This is the first report describing the regulation of an orphan RR by an HK. Moreover, this is also the first identification of cross-talk between different TCS components in S. coelicolor .     

Emergence and spread of carbapenem-resistant Acinetobacter baumannii in a tertiary care hospital in Turkey

An intensive care unit (ICU)-based OXA-23-producing multiple-drug resistant Acinetobacter baumannii (MDRAB) outbreak was detected between October 2005 and October 2006. A total of 47 patients were infected/colonized with the outbreak strain. Clinical data were available from 37 patients. The all-cause mortality rate among the patients exposed to the epidemic strain was 35% (13/37). The outbreak strain and the resistance determinants were characterized both by microbiological methods and by molecular techniques. Cloning and sequencing experiments identified ISAbaI-associated bla(oxa-23) on the chromosome. Screening of imipenem-resistant Acinetobacter isolated from the ICU during the outbreak period with PCR identified 97 isolates as positive for the ISAbaI-bla(oxa-23) structure. Pulsed-field gel electrophoresis and plasmid analyses with selected nonrepetitive isolates revealed the clonality. Disk diffusion on cloxacillin-supplemented agar media and the real-time PCR experiments showed that outbreak isolates are overexpressing the ampC enzyme. This study highlights the occurrence of OXA-23-producing and ampC-overexpressing MDRAB in ICUs.     

Distribution of blaOXA genes among carbapenem-resistant Acinetobacter baumannii nosocomial strains in Poland

Acinetobacter baumannii is an important nosocomial pathogen occurring particularly in intensive care (ICU) as well as burn therapy units (BTU). A. baumannii strains have emerged as resistant to almost all antimicrobial agents, including carbapenems. b-lactamase-mediated resistance is the most common mechanism for carbapenem resistance in this species. Carbapenem-hydrolysing class D b-lactamases - OXA are widespread among A. baumannii strains. It is suggested that ISAba1 plays an important role in drug resistance. The aims of the study were detection of OXA encoding genes and presence of ISAba1. The study included the total of 104 isolates of carbapenem-resistant A. baumannii, obtained from patients hospitalized in ICU and BTU of Specialized Hospital in Krakow. Multiplex PCR was applied for detection of selected OXA carbapenemases encoding genes. PCR analysis showed the presence of bla OXA-51-like gene and ISAba1 in all isolates. 46 strains carried bla OXA-51-like and bla OXA-23-like genes while 48 bla OXA-51-like and bla OXA-40-like genes. 3 isolates carried: bla OXA-51-like , bla OXA-23-like and bla OXA-40-like genes. 7 strains encoded an OXA-51-like carbapenemase but were negative for enzymes belonging to the other families tested. Comparative analysis of ICU and BTU isolates revealed the dominance of: bla OXA-51-like and bla OXA-40-like among ICU while bla OXA-51-like and bla OXA-23-like in BTU.     

Differential proteomics of the plasma of individuals with sepsis caused by Acinetobacter baumannii

This study examines alterations in the plasma proteome in ten adults affected by sepsis caused by Acinetobacter baumannii as compared to paired healthy controls. 2-DE profiles of plasma from patients and paired healthy donors, depleted of the six most abundant proteins, were analysed by the DIGE technique. Protein spot detection and quantification were performed with the Differential In-gel Analysis and Biological Variation Analysis modules of the DeCyder^™ software. Differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) after colloidal Coomassie blue staining.Almost 900 spots were detected on a unique 2-D gel by the DIGE technique. A total of 269 protein spots of differential abundance were shown to be statistically significant (2.5-fold) with p values of p ≤ 0.01 (135 spots) and p ≤ 0.05 (134 spots) as determined by the t test. Seventy-one spots were submitted to mass spectrometry and about 30% could be successfully identified.This multiplex approach significantly reduced experimental variability, allowing for the confident detection of small differences in protein levels. Results include differentially expressed lipoproteins as well as proteins belonging to inflammatory/coagulation pathways and the kallikrein–kinin system. These data improves the knowledge for future developments in sepsis diagnosis, staging and therapy.     

Proteomic analysis of a fraction enriched in cell envelope proteins of Acinetobacter baumannii

Acinetobacter baumannii is a multiresistant opportunistic nosocomial pathogen. A protein fraction was purified and analyzed by 2-DE. Twenty-nine major protein spots were selected for protein identification using trypsin digestion and MS analysis. As the A. baumannii genome has not yet been described, protein identification was performed by homology with other Acinetobacter species in the NCBi database. We identified ribosomal proteins, chaperones, elongation factors and outer membrane proteins (Omp), such as OmpA and the 33-36-kDa OMP. Proteomic analysis of A. baumannii provides a platform for further studies in antimicrobial resistance.     

Comparative genomics of multidrug resistance in Acinetobacter baumannii

Acinetobacter baumannii is a species of nonfermentative gram-negative bacteria commonly found in water and soil. This organism was susceptible to most antibiotics in the 1970s. It has now become a major cause of hospital-acquired infections worldwide due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents. Here we use a comparative genomic approach to identify the complete repertoire of resistance genes exhibited by the multidrug-resistant A. baumannii strain AYE, which is epidemic in France, as well as to investigate the mechanisms of their acquisition by comparison with the fully susceptible A. baumannii strain SDF, which is associated with human body lice. The assembly of the whole shotgun genome sequences of the strains AYE and SDF gave an estimated size of 3.9 and 3.2 Mb, respectively. A. baumannii strain AYE exhibits an 86-kb genomic region termed a resistance island—the largest identified to date—in which 45 resistance genes are clustered. At the homologous location, the SDF strain exhibits a 20 kb-genomic island flanked by transposases but devoid of resistance markers. Such a switching genomic structure might be a hotspot that could explain the rapid acquisition of resistance markers under antimicrobial pressure. Sequence similarity and phylogenetic analyses confirm that most of the resistance genes found in the A. baumannii strain AYE have been recently acquired from bacteria of the genera Pseudomonas, Salmonella, or Escherichia. This study also resulted in the discovery of 19 new putative resistance genes. Whole-genome sequencing appears to be a fast and efficient approach to the exhaustive identification of resistance genes in epidemic infectious agents of clinical significance.     

Comparative analysis of surface-exposed virulence factors of Acinetobacter baumannii

Background

Acinetobacter baumannii is a significant hospital pathogen, particularly due to the dissemination of highly multidrug resistant isolates. Genome data have revealed that A. baumannii is highly genetically diverse, which correlates with major variations seen at the phenotypic level. Thus far, comparative genomic studies have been aimed at identifying resistance determinants in A. baumannii. In this study, we extend and expand on these analyses to gain greater insight into the virulence factors across eight A. baumannii strains which are clonally, temporally and geographically distinct, and includes an isolate considered non-pathogenic and a community-acquired A. baumannii.

Results

We have identified a large number of genes in the A. baumannii genomes that are known to play a role in virulence in other pathogens, such as the recently studied proline-alanine-alanine-arginine (PAAR)-repeat domains of the type VI secretion systems. Not surprising, many virulence candidates appear to be part of the A. baumannii core genome of virulent isolates but were often found to be insertionally disrupted in the avirulent A. baumannii strain SDF. Our study also reveals that many known or putative virulence determinants are restricted to specific clonal lineages, which suggests that these virulence determinants may be crucial for the success of these widespread common clones. It has previously been suggested that the high level of intrinsic and adaptive resistance has enabled the widespread presence of A. baumannii in the hospital environment. This appears to have facilitated the expansion of its repertoire of virulence traits, as in general, the nosocomial strains in this study possess more virulence genes compared to the community-acquired isolate.

Conclusions

Major genetic variation in known or putative virulence factors was seen across the eight strains included in this study, suggesting that virulence mechanisms are complex and multifaceted in A. baumannii. Overall, these analyses increase our understanding of A. baumannii pathogenicity and will assist in future studies determining the significance of virulence factors within clonal lineages and/or across the species.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1020) contains supplementary material, which is available to authorized users.     

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).     

Persistence of carbapenem-resistant Acinetobacter baumannii strains in an Italian intensive care unit during a forty-six month study period

The aims of this study were to analyze carbapenem-resistance Acinetobacter baumannii isolates (CRAB) and their molecular epidemiology in an ICU of Southern Italy. Clinical outcomes and therapeutic management of patients are also described. The study was performed from January 2007 to October 2010. The presence of carbapenemases was determined by PCR. Strains were typed by PFGE. All A. baumannii isolates were carbapenem-resistant with imipenem MIC≥16 μg/mL. Molecular characterization showed the occurrence of a predominant clone. The most frequent infection by CRAB was ventilator-associated pneumonia; colistin was the drug of choice for this infection. The therapy was safe in all cases except in one where therapy was suspended due to the onset of acute renal failure. We documented the presence of CRAB in this ICU, besides the occurrence of a predominant clone, over all the study period. Despite the infection control procedures used, intra-facility A. baumannii transmission is evident as well as the significant capacity for long-term survival in the hospital environment.