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.     

Interspecies communication between Burkholderia cepacia and Pseudomonas aeruginosa

Burkholderia cepacia and Pseudomonas aeruginosa are opportunistic pathogens that commonly cause pulmonary infections in cystic fibrosis patients and occasionally co-infect patients' lungs. Both organisms possess quorum-sensing systems dependent on N-acyl homoserine lactone (N-acyl-HSL). Cross-feeding assays demonstrated that P. aeruginosa and B. cepacia were able to utilize heterologous N-acyl-HSL signaling molecules. The ability of quorum-sensing genes from one species to complement the respective quorum-sensing mutations in the heterologous species was also examined. These studies suggest that B. cepacia CepR can use N-acyl-HSLs synthesized by RhlI and LasI and that P. aeruginosa LasR and RhlR can use N-acyl-HSLs synthesized by CepI. It is possible that a mixed bacterial population of B. cepacia and P. aeruginosa can coordinately regulate some of their virulence factors and influence the progression of lung disease due to infection with these organisms.     

The identification of Pseudomonas cepacia and its occurrence in clinical material

During the 19 year period ending December 1984, 4840 strains of Gram-negative non-fermentative bacteria were submitted to the National Collection of Type Cultures for identification. Of these, 195 strains (4·0% of the total) were identified as Pseudomonas cepacia which demonstrates both that the species is regularly encountered in clinical material in the UK and that several laboratories have experienced difficulty in identifying the organism. The sources from which the 195 strains were isolated are reported and also the characteristics by which the species may be recognized. The clinical significance of Ps. cepacia is reviewed, and the resistance of this species to disinfectants and antimicrobial agents commonly used to treat pseudomonas infections is discussed to underline the necessity for the precise identification of Ps. cepacia.     

The identification of Pseudomonas cepacia and its occurrence in clinical material

During the 19 year period ending December 1984, 4840 strains of Gram-negative non-fermentative bacteria were submitted to the National Collection of Type Cultures for identification. Of these, 195 strains (4.0% of the total) were identified as Pseudomonas cepacia which demonstrates both that the species is regularly encountered in clinical material in the UK and that several laboratories have experienced difficulty in identifying the organism. The sources from which the 195 strains were isolated are reported and also the characteristics by which the species may be recognized. The clinical significance of Ps. cepacia is reviewed, and the resistance of this species to disinfectants and antimicrobial agents commonly used to treat pseudomonas infections is discussed to underline the necessity for the precise identification of Ps. cepacia.     

Degradation of 2-chlorobenzoate by Pseudomonas cepacia 2CBS

A bacterium was isolated from water by enrichment on 2-chlorobenzoate as sole source of carbon and energy. Based on morphological and physiological properties, this microorganism was assigned to the species Pseudomonas cepacia. The organism was designated Pseudomonas cepacia 2CBS. During growth on 2-chlorobenzoate, the chlorine substituent was released quantitatively, and a small amount of 2,3-dihydroxybenzoate accumulated in the culture medium. Mutants of Pseudomonas cepacia 2CBS were induced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Some of these mutants produced catechol from 2-chlorobenzoate. Other mutants accumulated the meta-cleavage product of catechol, 2-hydroxy-cis,cis-muconic acid semialdehyde. In crude cell-free extracts of Pseudomonas cepacia 2CBS, an enzyme was detected which catalysed the conversion of 2-chlorobenzoate to catechol. Molecular oxygen, NADH and exogenous Fe2+ were required for activity. Stoichiometric amounts of chloride were released. Experiments with 18O2 revealed that both oxygen atoms in the hydroxyl groups of the product were derived from molecular oxygen. Thus, the enzyme catalysing the conversion of 2-chlorobenzoate was identified as 2-chlorobenzoate 1,2-dioxygenase (1,2-hydroxylating, dehalogenating, decarboxylating). 2-Chlorobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS was shown to be a multicomponent enzyme system. The activities of catechol 2,3-dioxygenase and catechol 1,2-dioxygenase were detected in crude cell-free extracts. The activity of catechol 2,3-dioxygenase was 60 times higher than the activity of catechol 1,2-dioxygenase, indicating that catechol is mainly degraded via meta-cleavage in Pseudomonas cepacia 2CBS. No enzyme was found which converted 2,3-dihydroxybenzoate, suggesting that this compound is a dead-end metabolite of 2-chlorobenzoate catabolism. A pathway for the degradation of 2-chlorobenzoate by Pseudomonas cepacia 2CBS is proposed.     

Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia.

The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms. Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P. aeruginosa alone. The interactions between P. aeruginosa biofilms and B. cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship. Using the Calgary Biofilm Device (CBD), members of all genomovars of the B. cepacia complex were shown to form biofilms, including those isolated from CF lungs. Mixed species biofilm formation between CF isolates of P. aeruginosa and B. cepacia was readily achieved using the CBD. Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition. Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure. Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B. cepacia complex. Characterization of the soluble inhibitor suggested pyocyanin as the active compound.     

Burkholderia cepacia complex bacteria: opportunistic pathogens with important natural biology

Interaction with plants around their roots and foliage forms the natural habitat for a wide range of gram-negative bacteria such as Burkholderia, Pseudomonas and Ralstonia. During these interactions many of these bacteria facilitate highly beneficial processes such as the breakdown of pollutants or enhancement of crop growth. All these bacterial species are also capable of causing opportunistic infections in vulnerable individuals, especially people with cystic fibrosis (CF). Here we will review the current understanding of the Burkholderia cepacia complex (Bcc) as a group of model opportunistic pathogens, contrasting their clinical epidemiology with their ecological importance. Currently, the B. cepacia complex is composed of nine formally named species groups which are all difficult to identify using phenotypic methods. Genetic methods such as 16S rRNA and recA gene sequence analysis have proven useful for Bcc species identification. Multilocus sequence typing (MLST) is also emerging as a very useful tool for both Bcc strain and species identification. Historically, Burkholderia cenocepacia was the most dominant Bcc pathogen in CF, however, probably as a result of strict infection control practices introduced to control the spread of this species, its prevalence has been reduced. Burkholderia multivorans is the now the most dominant Bcc infection encountered in the UK CF population, a changing epidemiology that also appears to be occurring in the US CF population. The distribution of Bcc species residing in the natural environment may vary considerably with the type of environment examined. Clonally identical Bcc strains have been found to occur in the natural environment and cause infection. The contamination of medical devices, disinfectants and pharmaceutical formulations has also been directly linked to several outbreaks of infection. In the last 10 years considerable progress has been made in understanding the natural biology and clinical infections caused by this fascinating group of bacteria.     

Study of the effect of DNA polymorphisms in the mannose-binding lectin gene (MBL2) on disease severity in Slovak cystic fibrosis patients

Lung infections are the leading cause of morbidity and mortality in cystic fibrosis (CF). Mannose-binding lectin (MBL) is a key factor in innate immunity. We therefore investigated whether MBL2 gene variants are associated with pulmonary function or susceptibility to Pseudomonas aeruginosa and Burkholderia cepacia infection in Slovak patients affected with CF. DNA polymorphisms in exon 1 and the promoter region were typed by single base primer extension assay in 91 patients and 100 healthy controls. The concentrations of MBL protein were determined in 34 patients by a sandwich enzyme-linked immunosorbent assay, and spirometric and microbiological data were collected from medical records. In this study we found that MBL2 genotypes were associated neither with earlier acquisition of P. aeruginosa or B. cepacia nor with reduced pulmonary function among patients. Although MBL2 genotypes were associated with the MBL2 protein serum level, results were statistically significant only for polymorphisms in exon 1, with p = 0.0008. The role of the MBL2 gene in lung disease severity in CF patients represents a very complex phenomenon where both genetic and environmental factors play an important role in addition to that of the MBL2 gene. Understanding this complexity requires further studies based on a broader scale of genetic factors involving both a whole-genome approach and a larger patient cohort.     

Pseudomonas cepacia colonization and infection in intensive care units.

Pseudomonas cepacia has become a prominent epidemic nosocomial pathogen over the past 15 years. Between December 1982 and September 1983 it was isolated from 29 patients in two intensive care units (ICUs) at one hospital. Twelve infections--five bacteremias, four pneumonias and three urinary tract infections--occurred. Most of the isolates (25/29) were from the respiratory tract, and most (23/29) had the same antibiogram as the only environmental isolate, which was cultured from a contaminated ventilator thermometer, a previously unrecognized source of nosocomial infection. The ventilator thermometers were calibrated in a bath whose water had not been changed for months and contained P. cepacia. Despite elimination of this reservoir, P. cepacia was eradicated from the ICUs only after intensive infection control efforts were instituted.     

内毒素和降钙素原在妇科术后尿路感染中的鉴别诊断价值

目的评价中段尿内毒素和血清降钙素原在妇科术后不同种类细菌尿路感染中的鉴别诊断价值。方法收集临床1205例妇科术后患者中段尿进行细菌培养及内毒素检测,同时对患者进行血清降钙素原检测,比较结果对尿路感染的鉴别诊断价值。结果1205份标本中尿培养出阳性350例,感染率为29．04％,其中298例为均存在留置导尿管,而在剩余400例尿培养阴性的患者中仅仅120例留置导尿管。两组之间差异有统计学意义（χ2=26．78,P〈0．05）。其中革兰阴性杆菌189例（54％）,革兰阳性菌112例（32％）,真菌49例（14％）。在三组患者中,中段尿内毒素在革兰阴性菌引起的术后尿路感染较革兰阳性菌和真菌的患者中明显升高,差异均有统计学意义（P〈0．05）。而对于血清降钙素原在革兰阴性菌和革兰阳性菌感染的患者明显高于真菌尿路感染的患者,差异均有统计学意义（P〈0．05）。而在革兰阴性菌和革兰阳性菌感染的患者中差异无统计学意义（P〉0．05）。结论妇科术后尿路感染与留置导尿管密切相关,革兰阴性菌是引起妇科术后尿路感染的主要致病菌,中段尿内毒素有助于鉴别诊断出革兰阴性菌引起尿路感染,而血清PCT升高时则有助于排除真菌尿路感染。     

Rhodotorula infection. A systematic review of 128 cases from literature

Rhodotorula is an emerging opportunistic pathogen, particularly in immunocompromised patients. Many cases of fungemia associated with catheters, endocarditis, peritonitis, meningitis, and endophthalmitis are infections incited by this yeast. The main purpose of this study was to review all cases of Rhodotorula infection reported in the literature and to describe risk factors, underlying conditions and outcome. From 128 cases, 79% were fungemia (103 cases), 7% eye infections (nine cases) and 5% (six cases) peritonitis associated with continuous ambulatory peritoneal dialysis. Eighty seven percent of Rhodotorula infections are associated with underlying immunosuppression or cancer. The most common isolated risk factor associated with Rhodotorula infection was the use of a central venous catheter, which was found in 83.4% of Rhodotorula fungemia (86 cases). Rhodotorula mucilaginosa was the most common species of fungemia (74% of cases), followed by Rhodotorula glutinis with 7.7%. The species was not identified in 17% of the cases of fungemias. Amphotericin was the drug of choice in the treatment of fungemia and most of the eye infections were treated with topical amphotericin, although all patients lost their vision. All peritonitis cases associated with continous ambulatory peritoneal dialysis needed to have the Tenckoff catheter changed. The overall mortality of Rhodotorula infection was 12.6%.     

Comparative studies on the degradation of guanidino and ureido compounds by Pseudomonas

The utilization of guanidino and ureido compounds was studied in several Pseudomonas species. Multiple routes of agmatine catabolism were found. All members of the homology group I of Pseudomonas use the initial deamination of agmatine to carbamoylputrescine which is subsequently converted to putrescine. In Pseudomonas indigofera, the catabolism of agmatine can also occur via an initial hydrolysis of the amidino group to putrescine catalyzed by an agmatine amidinohydrolase. A third pathway was found in Pseudomonas cepacia, namely oxidative deamination producing guanidinobutyraldehyde catalyzed by agmatine dehydrogenase, followed by formation of guanidinobutyrate and removal of urea by guanidinobutyrate amidinohydrolase to produce 4-aminobutyrate. Novel amidino-hydrolases were characterized in P. putida for the utilization of arcaine and audouine, and in P. cepacia for arcaine, homoarginine and guanidinovalerate. Guanidinovalerate amidinohydrolase was also detected in P. doudoroffii. Some of these amidinohydrolases accept more than one substrate, e.g., guanidinobutyrate and guanidinovalerate utilization by P. doudoroffii and P. cepacia, the catabolism of arcaine and audouine by P. putida, and the degradation of arcaine and homoarginine by P. cepacia.     

A physical genome map of the Burkholderia cepacia type strain

Burkholderia cepacia (basonym Pseudomonas cepaci a), the type speciesof the new genus Burkholderia , is of interest, not only because of its broad catabolic capacity and its ability to antagonize soil-borne plant pathogens, but also because of its causative role in infections in man, which are particularly evident in patients with cystic fibrosis. A physical map of the 8.1 Mb genome of the B. cepacia type-strain ATCC 25416 was constructed by applying two-dimensional pulsed-field gel electrophoresis techniques. Placed onto the macrorestriction map were 38 Spel , 11 Swal , 11 Pacl , 11 Pmel and six l-Ceul sites, resulting in an average resolution of 1O5 kbp. Random single-hit linearization by irradiation and restriction mapping uncovered the presence of four circular replicons of 3.65 Mb, 3.17 Mb, 1.07 Mb and 200 kbp in size. The largest replicon harbours four rrn operons while the other two Megabase-size replicons each contain a single rrn operon, suggesting that the genome has three chromosomes and a large plasmid. Within the beta subdivision of proteobacteria, the existence of multiple replicons is not confined to B. cepacia . The phylogenetically related species Burkholderia glumae , Burkholderia pickettii , Burkholderia solanacearum , Alcaligenes eutrophus and the so far unassigned Pseudomonas glathei were also found to harbour more than one Megabase-size replicon.     

Cystic fibrosis: an inherited susceptibility to bacterial respiratory infections.

Cystic fibrosis is a severe monogenic disorder of ion transport in exocrine glands. The basic defect predisposes to chronic bacterial airway infections with Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa and Burkholderia cepacia. The Pseudomonas infections in cystic fibrosis are a paradigm of how versatile environmental bacteria can conquer, adapt and persist in an atypical habitat and successfully evade defence mechanisms and chemotherapy in a susceptible host. Regular chemotherapy with aerosol and systemic antipseudomonal drugs has improved the course and prognosis of the disease, and research for effective vaccines is on the way.     

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.     

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.     

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.     

Effects of Hydrogel/Silver Coatings on In Vitro Adhesion to Catheters of Bacteria Associated with Urinary Tract Infections

Sections of sterile all-silicone-, hydrogel/silver-all-silicone-, and hydrogel/silver-latex-Foley urinary catheters were exposed to suspensions of bacteria and Candida albicans associated with urinary tract infections. The adhesion of these microorganisms to the catheters was determined with a radiolabel–cell procedure and scanning electron microscopy. Anomalous data with the radiolabel procedure were produced with the hydrogel/silver-latex catheters for certain species. These aberrant data were related to adhesion on the untreated cut ends of the latex catheter. Radiolabel-cell-adhesion procedures that involve sections of coated materials may need to be supplemented with additional procedures such as scanning electron microscopy for valid interpretations of the data. Adhesion to the hydrogel/silver catheters by both Gram-positive- and Gram-negative bacteria most commonly associated with nosocomial urinary tract infections, including a strain of Pseudomonas aeruginosa noted for its superior adhesion capacity, was significantly lower than the adhesion to the control all-silicone catheter. Received: 21 January 2000 / Accepted: 26 February 2000     

泌尿外科住院患者泌尿系感染病原菌分布及其耐药性分析

了解泌尿外科住院患者泌尿系感染病原菌的分布及其对常用抗菌药物的耐药情况。对泌尿外科泌尿系感染住院患者的消毒中段尿培养结果进行回顾性分析,尿培养菌株的鉴定、药敏分析和统计分析采用VITEK2全自动微生物仪。3 a中泌尿外科泌尿系感染住院患者共分离到细菌1 233株,其中革兰阴性杆菌772株,占62.61%,革兰阳性球菌353株,占28.63%,真菌82株,占6.65%。菌株数居前5位的细菌依次为:大肠埃希菌、肠球菌属、洋葱伯克霍尔德菌、假丝酵母菌属和变形杆菌。分离大肠埃希菌产ESBLs率为66.18%,粪肠球菌中未发现VRE菌株,屎肠球菌VRE为0.8%。未发现对美洛培南耐药的大肠埃希菌,对肠埃希菌耐药率在10%以下的有亚胺培南、阿米卡星、哌拉西林/他唑巴坦和呋喃妥因。洋葱伯克霍尔德菌和铜绿假单胞菌对所监测的抗菌药物均有不同程度的耐药。未发现对万古霉素、替考拉宁及氨苄西林耐药的粪肠球菌。泌尿系感染的病原菌以大肠埃希菌和肠球菌为主,非发酵革兰阴性杆菌及假丝酵母菌所占比例超过20%,不容忽视,病原菌的耐药率较高,临床医生应根据尿培养和药敏试验结果合理使用抗菌药物。     

Transposon Tn5-259 mutagenesis of Pseudomonas cepacia to isolate mutants deficient in antifungal activity.

Transposon Tn5-259 was inserted into the chromosome of Pseudomonas cepacia by mating with an Escherichia coli strain harboring a self-mobilizable, temperature-sensitive plasmid, pME12. Data from Southern blots and auxotroph analyses indicated that a single copy of the transposon was inserted in several places into the chromosome of P. cepacia. Among 1500 Tn5-259 transconjugants, only one mutant was found to be defective in the production of an antifungal compound, pyrrolnitrin. In addition, this mutant lost its ability to antagonize fungal phytopathogens. Using flanking DNA of the mutated gene as a probe, we have isolated four overlapping cosmid clones from a genomic library of P. cepacia. However, we were unable to complement the mutant because of difficulty in mobilizing the cosmids from E. coli to P. cepacia.