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.     

Rapid differentiation of Pseudomonas pseudomallei from Pseudomonas cepacia

The Minitek disc system was utilized for the differentiation of Pseudomonas pseudomallei, the causative agent of melioidosis, from Ps. cepacia. The system was simple to use, inexpensive, and furnished rapid, clear-cut test results after 4 h. This procedure is suitable for differentiating soil bacteria presumptively identified as Ps. pseudomallei, Ps. cepacia or flavobacteria, and for the rapid confirmation of the presumptive identification of either Ps. pseudomallei or Ps. cepacia obtained by commercial identification-kit systems in the clinical laboratory.     

Phylogenetic studies of the rRNA group II pseudomonads based on 16S rRNA gene sequences

Nearly complete sequences of 16S rRNA genes were determined for eight bacterial strains representing five species of the rRNA homology group II pseudomonads that are members of the beta subclass of the class Proteobacteria. Comparative analysis with published sequence data indicated that Pseudomonas andropogonis, Ps. caryophylli, Ps. gladioli pv. gladioli and Ps. cepacia aggregate in one coherent cluster at 94·2% sequence similarity; Ps. solanacearum and Ps. pickettii shared 95·3% and 92·8% similarity with Alcaligenes eutrophus in another cluster. Both clusters joined at 87·8% similarity, which is similar to that for genera in this subclass of Proteobacteria. Based on this study and on comparison with other works we suggest that these species are separated from authentic pseudomonads and constitute a new genus or possibly two related genera accommodating Ps. andropogonis, Ps. caryophylli, Ps. gladioli, Ps. cepacia, and Ps. solanacearum, Ps. pickettii and A. eutrophus, respectively. Four strains of Ps. solanacearum representing Biovars 1, 2, 3 and 4 were subdivided into two clusters at 99·1% sequence similarity, in agreement with other published phenotypic and genotypic studies. The two clusters may be potentially regarded as subspecies.     

Numerical systematics of bacteria of the genus Pseudomonas

In 290 strains of bacteria belonging to the genus Pseudomonas, 120 morphological and physiologo-biochemical characters were studied and the results obtained thereby were analyzed by the methods of numerical taxonomy using computers. The majority of strains were subdivided into 11 clusters: Ps. aeruginosa (1), Ps. putida (2), Ps. rathonis (5), Ps. syringae (8), Ps. pseudoalcaligenes (9), Ps. maltophilia (10), Ps. acidovorans (11), Ps. testosteroni (12), Ps. mendocina (13), Ps. cepacia (14), Ps. fluorescens (3). The latter cluster included also the strains identified earlier as Ps. aurantiaca, Ps. lemonnieri, Ps. fluoro-violaceus, and Ps. aureofaciens. Three clusters contained strains which could not be identified and probably should be regarded as distinct species. The characteristics have been selected useful for diagnostics of the above Pseudomonas bacteria and the subgroups of Ps. fluorescens.     

Bacterial whole cell protein profiles of the rRNA group II pseudomonads

Studies on bacterial whole cell protein profiles showed that members of the rRNA group II pseudomonads were distinct from other non-fluorescent and fluorescent pseudomonads, including Pseudomonas aeruginosa, the type species of the genus Pseudomonas. Strains of Ps. andropogonis, Ps. caryophylli, Ps. gladioli pv. gladioli, Ps. pickettii, Ps. pseudomallei and Ps. rubrisubalbicans showed uniform and distinct protein patterns, while strains of Ps. solanacearum and Ps. cepacia displayed differences within species. Numerical analysis of their protein profiles with GelManager and Taxan programs generated dendrograms comprising 16 clusters at 89% similarity. Each cluster included strains belonging to the same species with the exception of Ps. solanacearum, which fragmented into three clusters. Pseudomonas solanacearum showed different protein patterns correlating with different biovars and the two divisions of Cook et al. (1989), as well as the results of 16S rRNA gene sequencing. The whole cell protein profiles of a total of 83 strains belonging to 14 bacterial species were numerically analysed.     

Identification and distribution of Pseudomonas stutzeri in clinical material

During the 19 year period ending December 1984, 114 (2.4%) of 4840 strains of Gram-negative non-fermentative bacteria submitted, mainly by laboratories in the UK, to the National Collection of Type Cultures for computer-assisted identification were strains of Pseudomonas stutzeri. These figures suggest that Ps. stutzeri is a relatively uncommon species in clinical material in the UK but that when it does occur laboratories have difficulty in identifying it. The sources from which the strains were isolated and also characteristics of the species by which it may be recognized are reported. The clinical significance of Ps. stutzeri is discussed and also the susceptibility of this species to antimicrobial agents.     

Identification and distribution of Pseudomonas stutzeri in clinical material

During the 19 year period ending December 1984, 114 (2.4%) of 4840 strains of Gram-negative non-fermentative bacteria submitted, mainly by laboratories in the UK, to the National Collection of Type Cultures for computer-assisted identification were strains of Pseudomonas stutzeri. These figures suggest that Ps. stutzeri is a relatively uncommon species in clinical material in the UK but that when it does occur laboratories have difficulty in identifying it. The sources from which the strains were isolated and also characteristics of the species by which it may be recognized are reported. The clinical significance of Ps. stutzeri is discussed and also the susceptibility of this species to antimicrobial agents.     

Development of a recA gene-based identification approach for the entire Burkholderia genus

Burkholderia is an important bacterial genus containing species of ecological, biotechnological, and pathogenic interest. With their taxonomy undergoing constant revision and the phenotypic similarity of several species, correct identification of Burkholderia is difficult. A genetic scheme based on the recA gene has greatly enhanced the identification of Burkholderia cepacia complex species. However, the PCR developed for the latter approach was limited by its specificity for the complex. By alignment of existing and novel Burkholderia recA sequences, we designed new PCR primers and evaluated their specificity by testing a representative panel of Burkholderia strains. PCR followed by restriction fragment length polymorphism analysis of an 869-bp portion of the Burkholderia recA gene was not sufficiently discriminatory. Nucleotide sequencing followed by phylogenetic analysis of this recA fragment differentiated both putative and known Burkholderia species and all members of the B. cepacia complex. In addition, it enabled the design of a Burkholderia genus-specific recA PCR that produced a 385-bp amplicon, the sequence of which was also able to discriminate all species examined. Phylogenetic analysis of 188 novel recA genes enabled clarification of the taxonomic position of several important Burkholderia strains and revealed the presence of four novel B. cepacia complex recA lineages. Although the recA phylogeny could not be used as a means to differentiate B. cepacia complex strains recovered from clinical infection versus the natural environment, it did facilitate the identification of clonal strain types of B. cepacia, B. stabilis, and B. ambifaria capable of residing in both niches.     

Characterization of Nonfermentative Nonfastidious Gram Negative Bacteria encountered in Medical Bacteriology

More than 90 morphological and physiological characters of 546 strains of nonfermentative, nonfastidious, Gram negative bacteria isolated from clinical specimens were examined to determine those features most useful for the identification of these bacteria. The species examined included Moraxella osloensis, Mor. lacunata, Acinetobacter anitratum, Ac. haemolyticus subsp. haemolyticus, Ac. haemolyticus subsp. alcaligenes, Ac. Iwoffi, Alcaligenes faecalis, Alc. odorans var. viridans, Pseudomonas fluorescens, Ps. putida, Ps. pseudomallei, Ps. maltophilia, Ps. stutzeri, Ps. acidovorans, Ps. alcaligenes , and atypical strains of Ps. aeruginosa.     

Identification of clinical isolates of Burkholderia cepacia and assessment of their pathogenicity in animal experiments

In experiments on animals study of pathogenicity of 9 clinical strains of Burkholderia cepacia isolated from patients with chronic lung diseases was performed. Preliminary identification of studied strains by means of biochemical and genetic methods allowed to establish their belonging to B. cepacia species. It was determined that 6 of 9 strains are epidemiologically significant. Experiments showed that bacteria of studied strains are not able to cause infectious process in white mice and hamadryas baboons. Conclusion about appropriateness of development and use of other biological models was made.     

Phenotypic and genotypic identification of bacteria from the Burkholderia cepacia complex

AIM: Evaluation of the diagnostic value of pheno- and genotypic characteristics of B. cepacia strains collection. MATERIALS AND METHODS: Phenotypic and genetic methods of identification and differentiation of 25 strains of the B. cepacia complex. RESULTS: Polyphasic taxonomic approach utilizing multiple diagnostic tests was used for accurate identification of Burkholderia species. Algorithm for identification of microorganisms from the B. cepacia complex was developed. CONCLUSION: Combined use of phenotypic and molecular genetic tests, such as recA gene PCR, is recommended for differentiation of the B. cepacia complex genomovars.     

Evaluation of preservative effectiveness in pharmaceutical products : the use of a wild strain of Pseudomonas cepacia

A sodium benzoate-sorbic acid preservative system of a pharmaceutical product was proved effective against a wild strain of Pseudomonas cepacia , following the official method of the Italian and British Pharmacopoeias. However, this preservative system was ineffective against a challenge of Ps. cepacia wild strain cells grown in the unpreserved pharmaceutical product and on culture media different from those described by the Pharmacopoeias. The adaptive resistance of the wild strain of Ps. cepacia was not demonstrated with a laboratory strain (ATCC 25609). In contrast, p- hydroxybenzoate-based preservative systems proved to be efficient in protecting the pharmaceutical product against a challenge of wild and laboratory strains of Ps. cepacia grown in the different conditions described above. The results obtained suggest the usefulness, in the official methods for testing pharmaceutical preservatives, of using wild microbial strains isolated from the pharmaceutical environment. Metabolic adaptive responses, capable of affecting the antimicrobial sensitivity of wild micro-organisms used to challenge the preserved product, can be detected by using cells grown in the unpreserved pharmaceutical product.     

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.     

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.     

Matrix-assisted laser desorption ionisation-time-of of-flight mass spectrometry of intact cells allows rapid identification of Burkholderia cepacia complex

The present study examined the potential of intact-cell matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid identification of Burkholderia cepacia complex (Bcc) bacteria using an Applied Biosystems 4700 Proteomics Analyser. Two software packages were used to analyse mass profiles based on densitometric curves and peak positions. The 75 strains examined, represented the nine established Bcc species and some commonly misidentified species, closely related or biochemically similar to Bcc and relevant in the context of cystic fibrosis microbiology. All Bcc strains clustered together, separated from non-Bcc strains. Within Bcc, most Bcc strains grouped in species specific clusters, except for Burkholderia anthina and Burkholderia pyrrocinia strains which constituted a single cluster. The present study demonstrates that MALDI-TOF MS is a powerful approach for the rapid identification of Bcc bacteria.     

Identification of Pseudomonas tolaasi: the White Line in Agar and Mushroom Tissue Block Rapid Pitting Tests

A sharply defined white line of precipitate forms in Pseudomonas Agar F (Difco) between the opaque white colonies of Pseudomonas tolaasi and translucent colonies of certain unidentified pseudomonads. This visible interaction has been utilized in a specific and reliable method for the identification of Ps. tolaasi. The white line test was positive when 113 isolates of Ps. tolaasi from five different countries were examined, whereas 154 isolates of pseudomonads other than Ps. tolaasi , including Ps. corrugata, Ps. delphinii, Ps. fluorescens, Ps. lachrymans, Ps. marginalis, Ps. pastinaceae, Ps. phaseolicola, Ps. aeruginosa, Ps. putida, Ps. syringae, Ps. mors-prunorum, Ps. cichorii, Ps. antirrhini, Ps. viridiflava, Ps. caryophylli, Ps. cepacia, Ps. mendocina, Ps. stutzeri, Ps. acidivorus and Ps. lemoignei did not give the white line reaction with a reacting translucent colony pseudomonad. Browning of mushrooms in host tests does not help in the identification of Ps. tolaasi , but a conspicuous pitting produced in less than 10 min at the cut surface of mushroom tissue is as specific as the white line test in detecting Ps. tolaasi in suspension in distilled water.     

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.     

Investigating Burkholderia cepacia complex populations recovered from Italian maize rhizosphere by multilocus sequence typing

The Burkholderia cepacia complex (BCC) comprises at least nine closely related species of abundant environmental microorganisms. Some of these species are highly spread in the rhizosphere of several crop plants, particularly of maize; additionally, as opportunistic pathogens, strains of the BCC are capable of colonizing humans. We have developed and validated a multilocus sequence typing (MLST) scheme for the BCC. Although widely applied to understand the epidemiology of bacterial pathogens, MLST has seen limited application to the population analysis of species residing in the natural environment; we describe its novel application to BCC populations within maize rhizospheres. 115 BCC isolates were recovered from the roots of different maize cultivars from three different Italian regions over a 9-year period (1994-2002). A total of 44 sequence types (STs) were found of which 41 were novel when compared with existing MLST data which encompassed a global database of 1000 clinical and environmental strains representing nearly 400 STs. In this study of rhizosphere isolates approximately 2.5 isolates per ST was found, comparable to that found for the whole BCC population. Multilocus sequence typing also resolved inaccuracies associated with previous identification of the maize isolates based on recA gene restriction fragment length polymorphims and species-specific polymerase chain reaction. The 115 maize isolates comprised the following BCC species groups, B. ambifaria (39%), BCC6 (29%), BCC5 (10%), B. pyrrocinia (8%), B. cenocepacia IIIB (7%) and B. cepacia (6%), with BCC5 and BCC6 potentially constituting novel species groups within the complex. Closely related clonal complexes of strains were identified within B. cepacia, B. cenocepacia IIIB, BCC5 and BCC6, with one of the BCC5 clonal complexes being distributed across all three sampling sites. Overall, our analysis demonstrates that the maize rhizosphere harbours a massive diversity of novel BCC STs, so that their addition to our global MLST database increased the ST diversity by 10%.     

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.     

Identification of Pseudomonas spp. Isolated from Milk Produced in South Eastern Queensland

S ummary . Bacteria were isolated from raw and pasteurized milks produced throughout S. E. Queensland. Milk samples were plated initially on penicillin agar or on milk agar and incubated at 30° and 7°, respectively. On the basis of primary characterization, 167 of the 330 isolates obtained were identified as Pseudomonas spp. The pseudomonads were further characterized in accordance with the taxonomic studies of the genus by Stanier, Palleroni & Doudoroff (1966). Species designations were ascribed to the Pseudomonas isolates on the basis of distinctive species characteristics in conjunction with similarity coefficients between each isolate and the ideal species phenotype, as follows: Ps. fluorescens (121), Ps. aeruginosa (16), Ps. putida (12), Ps. maltophilia (9), Ps. pseudoalcaligenes (5), Ps. cepacia (3) and Ps. alcaligenes (1). A dendrogram obtained by cluster analysis of the Pseudomonas isolates is included.