Plasmids of Pseudomonas cepacia strains of diverse origins.

Thirty-seven strains of Pseudomonas cepacia from clinical, pharmaceutical-industrial, and environmental origins were analyzed for the presence of plasmid DNA by a modification of the rapid alkaline extraction method of Birnboim (H. C. Birnboim, Methods Enzymol. 100:243-255, 1983). Plasmids were present in 31 strains (84%) from all sources, with no one source showing less than 75% plasmid carriage among its strains. The plasmid profiles indicated that the presence of large plasmids (146 to 222 kb) was the norm. Those strains with greater antibiotic resistance were mainly in the clinical and pharmaceutical groups and carried large plasmids (222 kb) that appeared essentially identical by restriction digest analysis. The ability for conjugative transfer was shown with the broad-host-range plasmid R751 carrying the gene for resistance to trimethoprim, one of the few antimicrobial agents effective against P. cepacia. The plasmid was transferred from Pseudomonas aeruginosa to P. cepacia strains as well as from P. cepacia transconjugants to other P. cepacia strains.     

Plasmids of Pseudomonas cepacia strains of diverse origins.

Thirty-seven strains of Pseudomonas cepacia from clinical, pharmaceutical-industrial, and environmental origins were analyzed for the presence of plasmid DNA by a modification of the rapid alkaline extraction method of Birnboim (H. C. Birnboim, Methods Enzymol. 100:243-255, 1983). Plasmids were present in 31 strains (84%) from all sources, with no one source showing less than 75% plasmid carriage among its strains. The plasmid profiles indicated that the presence of large plasmids (146 to 222 kb) was the norm. Those strains with greater antibiotic resistance were mainly in the clinical and pharmaceutical groups and carried large plasmids (222 kb) that appeared essentially identical by restriction digest analysis. The ability for conjugative transfer was shown with the broad-host-range plasmid R751 carrying the gene for resistance to trimethoprim, one of the few antimicrobial agents effective against P. cepacia. The plasmid was transferred from Pseudomonas aeruginosa to P. cepacia strains as well as from P. cepacia transconjugants to other P. cepacia strains.     

Biodegradation of synthetic and naturally occurring mixtures of mono-cyclic aromatic compounds present in olive mill wastewaters by two aerobic bacteria

Two bacterial strains, Ralstonia sp. LD35 and Pseudomonas putida DSM 1868, were assayed for their ability to degrade the monocyclic aromatic compounds commonly found in olive mill wastewaters (OMWs). The goal was to study the possibility of employing the two strains in the removal of these recalcitrant and toxic compounds from the effluents of anaerobic treatment plants fed with OMWs. At first, the two strains were separately assayed for their ability to degrade a synthetic mixture of nine aromatic acids present in OMWs, both in growing- and resting-cell conditions. Then, due to the complementary activity exhibited by the two strains, a co-culture of the two bacteria was tested under growing-cell conditions for degradation of the same synthetic mixture. Finally, the degradation activity of the co-culture on two fractions was studied. Both fractions one deriving from natural OMWs through reverse osmosis treatment and containing low-molecular weight organic molecules, and the other obtained from an anaerobic lab-scale treatment plant fed with OMWs, were rich in monocyclic aromatic compounds. The co-culture of the two strains was able to biodegrade seven of the nine components of the tested synthetic mix (2, 6-dihydroxybenzoic acid and 3, 4, 5-trimethoxybenzoic acid were the two undegraded compounds). In addition, an efficient biodegrading activity towards several aromatic molecules present in the two natural fractions was demonstrated.     

Identification and onion pathogenicity of Burkholderia cepacia complex isolates from the onion rhizosphere and onion field soil

Burkholderia cepacia complex strains are genetically related but phenotypically diverse organisms that are important opportunistic pathogens in patients with cystic fibrosis (CF,) as well as pathogens of onion and banana, colonizers of the rhizospheres of many plant species, and common inhabitants of bulk soil. Genotypic identification and pathogenicity characterization were performed on B. cepacia complex isolates from the rhizosphere of onion and organic soils in Michigan. A total of 3,798 putative B. cepacia complex isolates were recovered on Pseudomonas cepacia azelaic acid tryptamine and trypan blue tetracycline semiselective media during the 2004 growing season from six commercial onion fields located in two counties in Michigan. Putative B. cepacia complex isolates were identified by hybridization to a 16S rRNA gene probe, followed by duplex PCR using primers targeted to the 16S rRNA gene and recA sequences and restriction fragment length polymorphism analysis of the recA sequence. A total of 1,290 isolates, 980 rhizosphere and 310 soil isolates, were assigned to the species B. cepacia (160), B. cenocepacia (480), B. ambifaria (623), and B. pyrrocinia (27). The majority of isolates identified as B. cepacia (85%), B. cenocepacia (90%), and B. ambifaria (76%) were pathogenic in a detached onion bulb scale assay and caused symptoms of water soaking, maceration, and/or necrosis. A phylogenetic analysis of recA sequences from representative B. cepacia complex type and panel strains, along with isolates collected in this study, revealed that the B. cenocepacia isolates associated with onion grouped within the III-B lineage and that some strains were closely related to strain AU1054, which was isolated from a CF patient. This study revealed that multiple B. cepacia complex species colonize the onion rhizosphere and have the potential to cause sour skin rot disease of onion. In addition, the onion rhizosphere is a natural habitat and a potential environmental source of B. cenocepacia.     

Comparative Zone Electrophoresis of Enzymes of Pseudomonas solanacearum and Pseudomonas cepacia

The technique of starch-gel electrophoresis with specific staining for a series of enzymes was used to compare 21 Pseudomonas strains representing both P. cepacia and P. solanacearum. These experiments produced no evidence for close similarity of the two species. Twelve strains of P. solanacearum were compared by means of data obtained from nine different enzymes, and the data indicate that these strains belong in two biotypes. Except for the assignment of two strains, these groups are the same as the two major groups previously derived from nutritional properties and from deoxyribonucleic acid hybridization experiments. Eleven enzymes were available for comparisons of the P. cepacia strains. Eight of these strains form a homogeneous group, but the last strain, number 249, differs considerably from the other representatives of the species.     

Bacteriocin typing of Pseudomonas cepacia strains isolated from patients and the rhizosphere of plants

The work deals with the bacteriocin typing of 34 P. cepacia strains isolated from different sources with respect to both the capacity of synthesizing bactericins and sensitivity to them. The standard set of strains comprizing 8 P. cepacia bacteriocin-sensitive strains and 6 highly active cepaciacin producer strains was used. 24 P. cepacia strains belonged to 11 different S-types, 20 strains synthetized cepaciacins of new types.     

A selective medium for enumeration and recovery of Pseudomonas cepacia biotypes from soil.

TB-T medium provides a high degree of selectivity for and detection of Pseudomonas cepacia biotypes upon initial plating from soil. TB-T medium consists of a basal medium with glucose as the sole carbon source and asparagine as the sole nitrogen source. The selectivity of TB-T medium is based on the combination of trypan blue (TB) and tetracycline (T) (pH 5.5). On TB-T medium, 216 of 300 isolates (72%) from five different soil types were identified as P. cepacia. The remaining 28% were facultative organisms that could be separated readily from P. cepacia by anaerobic glucose fermentation and by their inability to grow at 41 degrees C. Molds were controlled on low soil dilutions by adding crystal violet, nystatin, or both. Elimination of either ingredient or elevation of the pH to 7.5 resulted in a pronounced loss of selectivity. The efficiency of recovery varied considerably among P. cepacia strains but was high enough for some strains (76 to 86%) to permit quantitative studies. TB-T medium combines a defined formulation with high selectivity and allows recovery of P. cepacia biotypes from low soil dilutions (10(1) to 10(3)).     

A quorum-quenching approach to investigate the conservation of quorum-sensing-regulated functions within the Burkholderia cepacia complex

Taxonomic studies of the past few years have shown that the Burkholderia cepacia complex, a heterogeneous group of B. cepacia-like organisms, consists of at least nine species. B. cepacia complex strains are ubiquitously distributed in nature and have been used for biocontrol, bioremediation, and plant growth promotion purposes. At the same time, B. cepacia complex strains have emerged as important opportunistic pathogens of humans, particularly those with cystic fibrosis. All B. cepacia complex species investigated thus far use quorum-sensing (QS) systems that rely on N-acylhomoserine lactone (AHL) signal molecules to express certain functions, including the production of extracellular proteases, swarming motility, biofilm formation, and pathogenicity, in a population-density-dependent manner. In this study we constructed a broad-host-range plasmid that allowed the heterologous expression of the Bacillus sp. strain 240B1 AiiA lactonase, which hydrolyzes the lactone ring of various AHL signal molecules, in all described B. cepacia complex species. We show that expression of AiiA abolished or greatly reduced the accumulation of AHL molecules in the culture supernatants of all tested B. cepacia complex strains. Phenotypic characterization of wild-type and transgenic strains revealed that protease production, swarming motility, biofilm formation, and Caenorhabditis elegans killing efficiency was regulated by AHL in the large majority of strains investigated.     

A selective medium for enumeration and recovery of Pseudomonas cepacia biotypes from soil

TB-T medium provides a high degree of selectivity for and detection of Pseudomonas cepacia biotypes upon initial plating from soil. TB-T medium consists of a basal medium with glucose as the sole carbon source and asparagine as the sole nitrogen source. The selectivity of TB-T medium is based on the combination of trypan blue (TB) and tetracycline (T) (pH 5.5). On TB-T medium, 216 of 300 isolates (72%) from five different soil types were identified as P. cepacia. The remaining 28% were facultative organisms that could be separated readily from P. cepacia by anaerobic glucose fermentation and by their inability to grow at 41 degrees C. Molds were controlled on low soil dilutions by adding crystal violet, nystatin, or both. Elimination of either ingredient or elevation of the pH to 7.5 resulted in a pronounced loss of selectivity. The efficiency of recovery varied considerably among P. cepacia strains but was high enough for some strains (76 to 86%) to permit quantitative studies. TB-T medium combines a defined formulation with high selectivity and allows recovery of P. cepacia biotypes from low soil dilutions (10(1) to 10(3)).     

Exopolysaccharide production by mucoid and non-mucoid strains of Burkholderia cepacia

Thirteen strains of Burkholderia cepacia from various origins with mucoid and non-mucoid phenotypes were assayed for exopolysaccharide (EPS) production. The EPS were characterized by glycosyl composition analysis and examination of the products resulting from lithium-ethylenediamine and Smith degradations. The results showed that all strains, including the non-mucoid strains, were able to produce EPS exhibiting the same structural features, i.e. presence of one rhamnosyl, three galactosyl, one mannosyl, one glucosyl and one glucuronosyl residues, suggesting that this EPS is representative of the B. cepacia species.     

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.     

Macromolecular properties of cepacian in water and in dimethylsulfoxide

Cepacian is the exopolysaccharide produced by the majority of the so far investigated clinical strains of the Burkholderia cepacia complex. This is a group of nine closely related bacterial species that might cause serious lung infections in cystic fibrosis patients, in some cases leading to death. In this paper the aggregation ability and the conformational properties of cepacian chain were investigated to understand its role in biofilm formation. Viscosity and atomic force microscopy studies in water and in mixed (dimethylsulfoxide/water) solvent indicated the formation of double stranded molecular structures in aqueous solutions. Inter-residue short distances along cepacian chain were investigated by NOE NMR, which showed that two side chains of cepacian were not conformationally free due to strong interactions with the polymer backbone. These interactions were attributed to hydrogen bonding and contributed to structure rigidity.     

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).     

Competition in chemostat culture between Pseudomonas strains that use different pathways for the degradation of toluene.

Pseudomonas putida mt-2, P. cepacia G4, P. mendocina KR1, and P. putida F1 degrade toluene through different pathways. In this study, we compared the competition behaviors of these strains in chemostat culture at a low growth rate (D = 0.05 h-1), with toluene as the sole source of carbon and energy. Either toluene or oxygen was growth limiting. Under toluene-limiting conditions, P. mendocina KR1, in which initial attack is by monooxygenation of the aromatic nucleus at the para position, outcompeted the other three strains. Under oxygen limitation, P. cepacia G4, which hydroxylates toluene in the ortho position, was the most competitive strain. P. putida mt-2, which metabolizes toluene via oxidation of the methyl group, was the least competitive strain under both growth conditions. The apparent superiority of strains carrying toluene degradation pathways that start degradation by hydroxylation of the aromatic nucleus was also found during competition experiments with pairs of strains of P. cepacia, P. fluorescence, and P. putida that were freshly isolated from contaminated soil.     

Burkholderia cepacia complex: distribution of genomovars among isolates from the maize rhizosphere in Italy

Burkholderia cepacia is a 'complex' in which seven genomic species or genomovars have so far been identified. It appears that all seven B. cepacia genomovars are capable of causing infections in vulnerable persons; in particular, the importance of Burkholderia multivorans (genomovar II) and B. cepacia genomovar III among cystic fibrosis isolates, especially epidemic ones, has been emphasized. In order to acquire a better comprehension of the genomovar composition of environmental populations of B. cepacia, 120 strains were isolated from the rhizosphere of maize plants cultivated in fields located in northern, central and southern Italy. The identification of the different genomovars was accomplished by a combination of molecular polymerase chain reaction (PCR)-based techniques, such as restriction fragment length polymorphism (RFLP) analysis of 16S rDNA (ARDRA), genomovar-specific PCR tests and RFLP analyses based on polymorphisms in the recA gene whole-cell protein electrophoresis. ARDRA analysis allowed us to distinguish between all B. cepacia genomovars except B. cepacia genomovar I, B. cepacia genomovar III and Burkholderia ambifaria (genomovar VII). The latter genomovars were differentiated by means of recA PCR tests and RFLP analyses. Among the rhizospheric isolates of B. cepacia, we found only B. cepacia genomovar I, B. cepacia genomovar III, Burkholderia vietnamiensis (genomovar V) and B. ambifaria. B. cepacia genomovars I and III and B. ambifaria were recovered from all three fields, whereas B. vietnamiensis was detected only in the population isolated from the field located in central Italy. Among strains isolated from northern and southern Italy, the most abundant genomovars were B. ambifaria and B. cepacia genomovar III respectively; in contrast, the population isolated in central Italy showed an even distribution of strains among genomovars. These results indicate that it is not possible to differentiate clinical and environmental strains, or pathogenic and non-pathogenic strains, of the B. cepacia complex simply on the basis of genomovar status, and that the environment may serve as a reservoir for B. cepacia genomovar III infections in vulnerable humans.     

Burkholderia cepacia complex genomovars: utilization of carbon sources, susceptibility to antimicrobial agents and growth on selective media

AIMS: To investigate the relationship between genomovar status and carbon source utilization, antibiotic susceptibility and growth ability on selective media of 142 clinical and environmental Burkholderia cepacia complex (Bcc) isolates belonging to all nine genomovars. METHODS AND RESULTS: Carbon source utilization and growth on selective media were tested by agar plate multipoint inoculation. Antimicrobial minimum inhibitory concentration (MIC) values were determined by agar dilution. Of all carbon sources, l-arabinose was most frequently utilized, supporting growth of 90% of all isolates. Burkholderia cepacia genomovar VI failed to utilize azelaic acid, penicillin G, phtalate, salicin and tryptamine. Overall, B. vietnamiensis and B. anthina were most susceptible and B. cepacia genomovar VI most resistant to antimicrobial agents. Burkholderia cepacia selective agar (BCSA) and the Mast B. cepacia medium supported growth of Bcc isolates most efficiently. CONCLUSIONS: This study demonstrates phenotypic heterogeneity within the Bcc. Some trends can be observed at the genomovar level, but only B. cepacia genomovar VI could be differentiated unambiguously on the basis of its inability to grow on PCAT. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides an update on some differential phenotypic characteristics of all nine Bcc genomovars.     

从植物根际定向批量筛选广谱有机溶剂耐受性脂肪酶产生菌

洋葱伯克霍尔德菌脂肪酶是一类具有重要工业应用价值的优良脂肪酶之一。根据已公布的洋葱伯克霍尔德菌基因组信息, 在传统的洋葱伯克霍尔德菌选择性培养基中添加适量的氨苄青霉素和卡那霉素, 从植物根际的土壤中筛选洋葱伯克霍尔德菌。对获得的单菌落再用含罗丹明B指示剂的产脂肪酶定性检测平板检测, 从4个根际土壤中筛选到35株产脂肪酶的洋葱伯克霍尔德菌, 阳性率达到65%。其中15株对体积浓度为10%的苯、己烷和正庚烷同时具有耐受性。用recA基因分子鉴定上述15株菌种, 全部属于洋葱伯克霍尔德菌菌群。     

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.     

Bacterial metabolism of 3-chloroacrylic acid.

Two bacterial strains were isolated with 3-chloroacrylic acid (CAA) as sole source of carbon and energy. Strain CAA1, a Pseudomonas cepacia sp., was capable of growth with only the cis-isomer of CAA. Strain CAA2, a coryneform bacterium, utilized both isomers of CAA as sole source of carbon and energy. Strain CAA1 contained cis-CAA hydratase and strain CAA2 contained two hydratases, one with cis-CAA hydratase activity and one with trans-CAA hydratase activity. The product of the hydratase activities with CAA was malonate semialdehyde. In both strains malonate semialdehyde was subsequently decarboxylated by a cofactor-independent decarboxylase yielding acetaldehyde and CO2.