Serum sensitivity of Burkholderia (Pseudomonas) cepacia isolates from patients with cystic fibrosis

Abstract Bacterial strains which are sensitive to the bactericidal activity of serum are generally considered to be less virulent than serum-resistant strains and are seldom associated with bacteraemia. Burkholderia ( Pseudomonas ) cepacia is an important pathogen in cystic fibrosis and is associated with rapid fatal pulmonary decline and bacteraemia in 20% of colonised patients. In this study 19 isolates of B. cepacia expressing either rough or smooth LPS were investigated to determine the degree of serum sensitivity. Strains expressing rough-LPS were serum-sensitive: these included a highly transmissible strain of B. cepacia isolated from approximately 50 cystic fibrosis patients attending various U.K. regional centres and associated with cases of bacteraemia.     

Type 1 and type 2 responses to Leishmania major

Pseudomonas aeruginosa and Burkholderia cepacia cause destructive lung disease in cystic fibrosis (CF) patients. Both pathogens employ 'quorum sensing', i.e. cell-to-cell communication, via diffusible N-acyl-L-homoserine lactone (AHL) signal molecules, to regulate the production of a number of virulence determinants in vitro. However, to date, evidence that quorum sensing systems are functional and play a role in vivo is lacking. This study presents the first direct evidence for the presence of AHLs in CF sputum. A total of 42 samples from 25 CF patients were analysed using lux-based Escherichia coli AHL biosensors. AHLs were detected in sputum from patients colonised by P. aeruginosa or B. cepacia but not Staphylococcus aureus. Furthermore, using liquid chromatography-mass spectrometry and thin layer chromatography, we confirmed the presence of N-hexanoylhomoserine lactone and N-(3-oxododecanoyl)homoserine lactone respectively in sputum samples from patients colonised by P. aeruginosa.     

Serum IgG response to Burkholderia cepacia outer membrane antigens in cystic fibrosis: Assessment of cross-reactivity with Pseudomonas aeruginosa

Abstract Burkholderia cepacia (Pseudomonas cepacia) is now recognised as an important pathogen in cystic fibrosis patients, and several reports have suggested that sputum-culture-proven colonisation occurs despite the presence of specific antibody. In an attempt to establish the use of antibody studies as diagnostic and prognostic indicators of B. cepacia infection, we have examined the IgG response to B. cepacia outer membrane proteins and lipopolysaccharide in patients also colonised with P. aeruginosa . The B. cepacia strains were grown in a modified iron-depleted chemically defined medium and outer membrane components examined by SDS-PAGE and immunoblotting. IgG antibodies were detected against B. cepacia outer membrane antigens, which were not diminished by extensive preadsorption with P. aeruginosa . The response to B. cepacia O-antigen could be readily removed by adsorption of serum either with B. cepacia whole cells or purified LPS, whereas we were unable to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells. The inability to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells maybe due to non-exposed surface epitopes. Several B. cepacia sputum-culture negative patients colonised with P. aeruginosa had antibodies directed against B. cepacia outer membrane protein. This study suggests that there is a specific anti- B. cepacia LPS IgG response, which is not due to antibodies cross-reactive with P. aeruginosa . Our studies indicate that much of the B. cepacia anti-outer membrane protein response is specific and not attributable to reactivity against co-migrating LPS.     

Defining potential targets for immunotherapy in Burkholderia cepacia infection

Abstract Burkholderia cepacia has become a serious source of infection in patients with cystic fibrosis. Antibiotic therapy is difficult as the bacteria are intrinsically resistant to most antibiotics. The present study compared the antibody response by immunoblot of 50 negative control sera, 22 patients with cystic fibrosis and no evidence of B. cepacia , 9 clinically well patients with cystic fibrosis colonised by B. cepacia and 5 patients with cystic fibrosis and deteriorating or fatal B. cepacia infection. Nineteen antigenic bands varying in apparent molecular weights from 19 to 170 kDa were identified. Two bands at 19 and 21 kDa were only present when the organism was grown in an iron-deficient medium. The band at 30 kDa was identified as a porin and the possession of IgG antibody carried a statistically significant ( P = 0.00003) better prognosis. This antigen was thus a potential target for immunotherapy.     

Lack of correlation between O-serotype,bacteriophage susceptibility and genomovar status in the Burkholderia cepacia complex

The Burkholderia cepacia complex comprises at least nine phylogenetically related genomic species (genomovars) which cause life-threatening infection in immunocompromised humans, particularly individuals with cystic fibrosis or chronic granulomatous disease. Prior to recognition that 'B. cepacia' comprise multiple species, in vitro studies revealed that the lipopolysaccharide (LPS) of these Gram-negative bacteria is strongly endotoxic. In this study, we used 117 B. cepacia complex isolates to determine if there is a correlation between O-antigen serotype and genomovar status. Isolates were also tested for their ability to act as bacterial hosts for the LPS-binding bacteriophages NS1 and NS2. The absence of genomovar II (Burkholderia multivorans) in 'historical B. cepacia' isolates was notable. Neither O-serotype nor phage susceptibility correlated with genomovar status. We conclude that variability in LPS may contribute to the success of these highly adaptable bacteria as human pathogens.     

Evidence of transmission of Burkholderia cepacia, Burkholderia multivorans and Burkholderia dolosa among persons with cystic fibrosis

Previous studies have identified specific Burkholderia cepacia complex strains that are common to multiple persons with cystic fibrosis (CF). Such so-called epidemic strains have an apparent enhanced capacity for inter-patient spread and reside primarily in Burkholderia cenocepacia (formerly B. cepacia complex genomovar III). We sought to identify strains from B. cepacia complex species other than B. cenocepacia that are similarly shared by multiple CF patients. We performed genotype analysis of 360 recent sputum culture isolates from 360 persons residing in 29 cities by using repetitive extragenic palendromic polymerase chain reaction (rep-PCR) and pulsed field gel electrophoresis. The results indicate that sharing of a common Burkholderia multivorans strain occurs relatively infrequently; however, several small clusters of patients infected with the same strain were identified. A cluster of seven patients infected with the same B. cepacia (genomovar I) strain was found. We also identified a large group of 28 patients receiving care in the same treatment center and infected with the same Burkholderia dolosa strain. These observations suggest that B. cepacia complex strains in species other than B. cenocepacia may be spread among CF patients.     

Complete structural characterization of the lipid A fraction of a clinical strain of B. cepacia genomovar I lipopolysaccharide

Burkholderia cepacia, a Gram-negative bacterium ubiquitous in the environment, is a plant pathogen causing soft rot of onions. This microorganism has recently emerged as a life-threatening multiresistant pathogen in cystic fibrosis patients. An important virulence factor of B. cepacia is the lipopolysaccharide (LPS) fraction. Clinical isolates and environmental strains possess LPS of high inflammatory nature, which induces a high level production of cytokines. For the first time, the complete structure of the lipid A components isolated from the lipopolysaccharide fraction of a clinical strain of B. cepacia is described. The structural studies carried out by selective chemical degradations, MS, and NMR spectroscopy revealed multiple species differing in the acylation and in the phosphorylation patterns. The highest mass species was identified as a penta-acylated tetrasaccharide backbone containing two phosphoryl-arabinosamine residues in addition to the archetypal glucosamine disaccharide [Arap4N-l-beta-1-P-4-beta-D-GlcpN-(1-6)-alpha-D-GlcpN-1-P-1-beta-L-Arap4N]. Lipid A fatty acids substitution was also deduced, with two 3-hydroxytetradecanoic acids 14:0 (3-OH) in ester linkage, and two 3-hydroxyhexadecanoic acids 16:0 (3-OH) in amide linkage, one of which was substituted by a secondary 14:0 residue at its C-3. Other lipid A species present in the mixture and exhibiting lower molecular weight lacked one or both beta-L-Arap4N residues.     

Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.

Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF). This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P. aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B. cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism. While understanding the mechanism of conversion to mucoidy in P. aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B. cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity.     

Incidence of the cblA major subunit pilin gene amongst Burkholderia species

PCR-amplification has been used to screen 75 isolates of the Burkholderia cepacia complex for the cblA pilin gene. PCR-amplified products of the correct size (664 bp) were cloned and sequenced and the sequences compared. Apart from in the control, epidemic cystic fibrosis (CF)-associated B. cepacia lineage we also identified, for the first time, cblA genes in a unique, non-CF clinical isolate from France and a plant (onion) pathogenic isolate from Italy. The sequence of the cblA gene amplified from the clinical isolate was more diverged from the epidemic lineage than that amplified from the onion pathogenic isolate.     

Highly purified lipopolysaccharides from Burkholderia cepacia complex clinical isolates induce inflammatory cytokine responses via TLR4-mediated MAPK signalling pathways and activation of NFkappaB

In cystic fibrosis (CF), bacteria of the Burkholderia cepacia complex (Bcc) can induce a fulminant inflammation with pneumonitis and sepsis. Lipopolysaccharide (LPS) may be an important virulence factor associated with this decline but little is known about the molecular pathogenesis of Bcc LPS. In this study we have investigated the inflammatory response to highly purified LPS from different Bcc clinical isolates and the cellular signalling pathways employed. The inflammatory response (TNFalpha, IL-6) was measured in human MonoMac 6 monocytes and inhibition experiments were used to investigate the Toll-like receptors and associated adaptor molecules and pathways utilized. LPS from all clinical Bcc isolates induced significant pro-inflammatory cytokines and utilized TLR4 and CD14 to mediate activation of mitogen-activated protein kinase pathways, IkappaB-alpha degradation and NFkappaB activation. However, LPS from different clinical isolates of the same clonal strain of Burkholderia cenocepacia were found to induce a varied inflammatory response. LPS from clinical isolates of Burkholderia multivorans was found to activate the inflammatory response via MyD88-independent pathways. This study suggests that LPS alone from clinical isolates of Bcc is an important virulence factor in CF and utilizes TLR4-mediated signalling pathways to induce a significant inflammatory response.     

Sensitivity of the Burkholderia cepacia complex and Pseudomonas aeruginosa to transducing bacteriophages

Burkholderia cepacia is now recognised as a life-threatening pathogen among several groups of immunocompromised patients. In this context, the proposed large-scale use of these bacteria in agriculture has increased the need for a better understanding of the genetics of the species forming the B. cepacia complex. Until now, little information has been available on the bacteriophages of the B. cepacia complex. Transducing phages, named NS1 and NS2, were derived from the lysogenic B. cepacia strains ATCC 29424 and ATCC 17616. The frequency of transduction per phage particle ranged from 1.0x10(-8) to 7.0x10(-6) depending on the phage and recipient strain used. The host range of NS1 and NS2 differed but in each case included environmental and clinical isolates, and strains belonging to several species and genomovars of the B. cepacia complex. The host range of both phages also included Pseudomonas aeruginosa. Some B. cepacia complex isolates were sensitive to the well-characterised P. aeruginosa transducing phages, B3, F116L and G101. The lytic activity of NS1 and NS2 was inhibited by B. cepacia lipopolysaccharide suggesting that this moiety is a binding site for both phages. The molecular size of the NS1 and NS2 genomes was approximately 48 kb.     

A novel strategy for the isolation and identification of environmental Burkholderia cepacia complex bacteria

The purpose of this study was to develop a novel strategy for the isolation and identification of Burkholderia cepacia complex bacteria from the home environment of cystic fibrosis (CF) patients. Water and soil samples were enriched in a broth containing 0.1% l-arabinose, 0.1% l-threonine, and a mixture of selective agents including 1 microgml(-1) C-390, 600U ml(-1) polymyxin B sulfate, 10 microgml(-1) gentamycin, 2 microgml(-1) vancomycin and 10 microgml(-1) cycloheximide. On selective media (consisting of the same components as above plus 1.8% agar), several dilutions of the enrichment broth were inoculated and incubated for 5 days at 28 degrees C. Isolates with different randomly amplified polymorphic DNA patterns were inoculated in Stewart's medium. Putative B. cepacia complex bacteria were confirmed by means of recA PCR and further identified by HaeIII-recA restriction fragment length polymorphism analysis. Our results suggest that these organisms may be more widespread in the home environment than previously assumed and that plant associated soil and pond water may be reservoirs of B. cepacia complex infection in CF patients.     

PCR-based identification and characterization of Burkholderia cepacia complex bacteria from clinical and environmental sources

AIMS: To study the genotypic identification and characterization of the 119 Burkholderia cepacia complex (Bcc) strains recovered from clinical and environmental sources in Japan and Thailand. METHODS AND RESULTS: Based on the results of analysis by 16S rDNA RFLP generated after digestion with DdeI, the Bcc strains were differentiated into two patterns: pattern 1 (including Burkholderia vietnamiensis) and pattern 2 (including B. cepacia genomovar I, Burkholderia cenocepacia and Burkholderia stabilis). All strains belonged to pattern 2 except for one strain. In the RFLP analysis of the recA gene using HaeIII, strains were separated into eight patterns designated as A, D, E, G, H, I, J and K, of which pattern K was new. Burkholderia cepacia epidemic strain marker (BCESM) encoded by esmR [corrected] and the pyrrolnitrin biosynthetic locus encoded by prnC were present in 22 strains (18%) and 88 strains (74%) from all sources, respectively. All esmR-positive [corrected] strains belonged to B. cenocepacia, whereas most prnC-positive strains belonged to B. cepacia genomovar I. CONCLUSIONS: Strains derived from clinical sources were assigned to B. cepacia genomovar I, B. cenocepacia, B. stabilis and B. vietnamiensis. The majority of Bcc strains from environmental sources (77 of a total 95 strains) belonged to B. cepacia genomovar I, whereas the rest belonged to B. cenocepacia. On the basis of genomovar-specific PCR and prnC RFLP analysis, strains belonging to recA pattern K were identified as B. cepacia genomovar I. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the genotypic identification of a collection of the Bcc strains from Japan and Thailand. RFLP analysis of the prnC gene promises to be a useful method for differentiating Burkholderia pyrrocinia from B. cepacia genomovar I strains.     

洋葱伯克霍尔德菌CF_66抗菌物质的分离纯化及性质的研究

洋葱伯克霍尔德菌CF-66能够抑制立枯丝核菌等若干植物病原菌和其它一些真菌的生长。CF-66菌发酵液的粗提液通过Sephadex-75pg、Sephacryl S-100柱层析分离纯化,获得抗菌物质CF66I。此抗菌物质耐热性强,耐碱,但在强酸性条件下不稳定。低浓度有机溶剂的存在有利于抑菌活性的提高。对其结构的研究表明CF66I是以(CH2CH2O)n为主要单元结构并带有酰氨键的化合物。     

Pro-inflammatory effects of Burkholderia cepacia on cystic fibrosis respiratory epithelium

Burkholderia cepacia causes pulmonary infection with high mortality in cystic fibrosis (CF) patients which is likely to involve interaction with respiratory epithelium. In this study the pro-inflammatory properties of B. cepacia were examined using a range of respiratory epithelial cell lines. B. cepacia and cell-free culture supernatants were used to stimulate cell lines with (SigmaCFTE29o- and IB3) and without (A549) the CF transmembrane conductance regulator mutation (CFTR), together with corrected cell lines (C38 and S9). Interleukin (IL)-6 and IL-8, but not GM-CSF or IL-1beta, were released from all the cell lines whereas PGE(2) (prostaglandin E(2)) was released from the A549, IB3 and S9 cell lines only. Nuclear factor (NF)-kappaB activation preceded cytokine release and suppression of NF-kappaB activity diminished cytokine release. These studies indicated that B. cepacia secretory products are potent pro-inflammatory agents for respiratory epithelium and suggest functional CFTR is not required for cytokine or prostanoid responses.     

重症监护病房洋葱伯克霍尔德菌医院感染的特征及耐药性分析

目的了解重症监护病房（ICU）洋葱伯克霍尔德菌引起医院感染的特征及耐药情况,为临床治疗及控制该菌的暴发流行提供实验依据。方法常规方法对我院2003年1月至2007年10月ICU的病人的各种临床标本进行分离培养,细菌鉴定及药敏试验采用全自动微生物鉴定仪VITEK-2进行。结果引起ICU医院感染的洋葱伯克霍尔德菌共有99例,感染以肺部感染为主,对临床常用的多种抗菌药物表现交叉耐药,对头孢匹肟、亚胺培南、哌拉西林、阿米卡星、庆大霉素的敏感率较差在50．0％以下;对环丙沙星、左氧氟沙星、头孢他啶、氨曲南、哌拉西林／他唑巴坦、美诺培南和复方新诺明的敏感率较高,分别为82．8％、87．9％、91．9％、72．7％、55．6％、62．6％和100．0％。结论引起ICU医院感染的洋葱伯克霍尔德菌具有多重耐药性,临床治疗时应根据药敏结果选用抗菌药物。     

Susceptibility of Burkholderia cepacia and other pathogens of importance in cystic fibrosis to u.v. light

To investigate the potential usefulness of u.v. germicidal irradiation (UVGI) in preventing the spread of Burkholderia cepacia, an important pathogen in cystic fibrosis (CF), the in-vitro susceptibility of B. cepacia to UVGI was determined. Five strains were exposed to UVGI from a 7.2-W source. Burkholderia cepacia was less susceptible to UVGI than other important CF-related pathogens, namely Staphylococcus aureus and Pseudomonas aeruginosa, but was more susceptible than Stenotrophomonas maltophilia. No strain of B. cepacia survived longer than an 8 s exposure to UVGI, with doses required to achieve 1 log reduction in bacterial numbers ranging from 28.3 to 57.5 J m(-2).     

洋葱伯克霍尔德菌致恶性肿瘤患者医院获得性感染的耐药性分析

目的分析我院恶性肿瘤患者医院获得性感染洋葱伯克霍尔德菌的临床特点及对常用抗菌药物的耐药性,为临床合理治疗提供依据。方法回顾性分析2011年1月至2013年12月,从我院恶性肿瘤感染患者送检的细菌培养标本;细菌鉴定用美国BD公司phoenix-100全自动细菌鉴定药敏系统,药敏试验采用纸片法,同时使用WHONET 5.6软件对相关资料进行统计。结果从检测部位分析主要分布在下呼吸道（74.1%）,其次为血液（9.4%）;药敏试验表明139株洋葱伯克霍尔德菌对米诺环素、氯霉素、美罗培南、头孢他啶和头孢哌酮/舒巴坦仍较敏感,可作为临床治疗洋葱伯克霍尔德菌感染的首选药物,其余15种抗菌药物的耐药率高达30.0%~100%。结论洋葱伯克霍尔德菌在恶性肿瘤患者中的耐药现象非常严重,临床应引起高度关注,及早进行微生物学检测,并根据药敏试验结果合理选用抗菌药物。     

Activation of MMP-9 by human lung epithelial cells in response to the cystic fibrosis-associated pathogen Burkholderia cenocepacia reduced wound healing in vitro

Burkholderia cepacia complex is a group of bacterial pathogens that cause opportunistic infections in cystic fibrosis (CF). The most virulent of these is Burkholderia cenocepacia. Matrix metalloproteinases (MMPs) are upregulated in CF patients. The aim of this work was to examine the role of MMPs in the pathogenesis of B. cepacia complex, which has not been explored to date. Real-time PCR analysis showed that B. cenocepacia infection upregulated MMP-2 and MMP-9 genes in the CF lung cell line CFBE41o- within 1 h, whereas MMP-2, -7, and -9 genes were upregulated in the non-CF lung cell line 16HBE14o-. Conditioned media from both cell lines showed increased MMP-9 activation following B. cenocepacia infection. Conditioned media from B. cenocepacia-infected cells significantly reduced the rate of wound healing in confluent lung epithelia (P < 0.05), in contrast to conditioned media from Pseudomonas aeruginosa-infected cells, which showed predominant MMP-2 activation. Treatment of control conditioned media from both cell lines with the MMP activator 4-aminophenylmercuric acetate (APMA) also resulted in clear activation of MMP-9 and to a much lesser extent MMP-2. APMA treatment of control media also delayed the repair of wound healing in confluent epithelial cells. Furthermore, specific inhibition of MMP-9 in medium from cells exposed to B. cenocepacia completely reversed the delay in wound repair. These data suggest that MMP-9 plays a role in the reduced epithelial repair observed in response to B. cenocepacia infection and that its activation following B. cenocepacia infection contributes to the pathogenesis of this virulent pathogen.     

Cystic fibrosis infections: treatment strategies and prospects

Pseudomonas aeruginosa and Burkholderia cepacia are the two major Gram-negative rods that colonize/infect the lungs of patients with cystic fibrosis (CF). These organisms may cause progressive respiratory failure, although occasionally more rapid infections result in the ' Cepacia ' syndrome. Many antibiotics have been used against Pseudomonas and Burkholderia , but once chronic colonization has been established, eradication of these organisms is rare. Drug therapy for CF patients is compromised by a number of bacterial factors that render the infectious agents resistant to antibiotics, including efflux pumps that remove antibiotics, lack of penetration of antibiotics into bacterial biofilms, and changes in the cell envelope that reduce the permeability of antibiotics. Any combination of these mechanisms increases the likelihood of bacterial survival. Therefore, combinations of antibiotics or of antibiotic and nonantibiotic compounds are currently being tested against Pseudomonas and Burkholderia . However, progress has been slow, with only occasional combinations showing promise for the eradication of persistent Gram-negative rods in the airways of CF patients. This review will summarize the current knowledge of CF infections and speculate on potential future pathways to treat these chronic infections.