The serological characteristics of the O antigens of Pseudomonas aeruginosa isolated from patients with a Pseudomonas infection during immunotherapy]

Serologic characteristics of P. aeruginosa O-antigens isolated from patients with P. aeruginosa infection were studied over the course of treatment with anti-P. aeruginosa sheep immunoglobulin. The preparation was used in 54 patients with nongeneralized forms of P. aeruginosa infection (infected wounds, pleural empyema) externally or intraperitoneally. From the clinical material collected from the patients a total of 54 P. aeruginosa strains were isolated. Serologic typing of the isolated strains with factor or group diagnostic agglutinating sera has revealed the O-group composition of the isolated strains; 66% of them were classified with O-groups 2,3, and 6. Serologic variants of the strains isolated from patients proved to be stable over the course of the disease immunotherapy. Analysis of the results of bacteriologic control of immunotherapy. efficacy and the clinical data has demonstrated the efficacy of immunotherapy in 61.1% of cases and its partial effect in 20.4% of cases of P. aeruginosa infection.     

Characteristics of hospital strains of Pseudomonas aeruginosa

A total of 745 P. aeruginosa strains from patients with purulent inflammatory processes, 216 strains from the environment of a surgical hospital and 35 strains from carriers were studied with respect to 30 cultural and biochemical signs of P. aeruginosa. 19.8% of the strains were found to form no pigment, and in 14.8% of the strains delayed pigment formation was observed (on days 3-10). The most stable signs were motility (99.6%), growth in Simmons citrate agar (97.6%), growth at 42 degrees C (97.4%), arginine decarboxylase activity (96.8%). In 77.0% of the strains glucose assimilation in Hiss liquid medium, in 85.6% glucose oxidation in the OF test, in 90.8% the formation of urease and in 93.2% the formation of gelatinase were observed. Among the strains isolated from the environment, P. aeruginosa variants, atypical with respect to their main differentiating signs, were isolated significantly more frequently.     

Rhizosphere selection of highly motile phenotypic variants of Pseudomonas fluorescens with enhanced competitive colonization ability

Phenotypic variants of Pseudomonas fluorescens F113 showing a translucent and diffuse colony morphology show enhanced colonization of the alfalfa rhizosphere. We have previously shown that in the biocontrol agent P. fluorescens F113, phenotypic variation is mediated by the activity of two site-specific recombinases, Sss and XerD. By overexpressing the genes encoding either of the recombinases, we have now generated a large number of variants (mutants) after selection either by prolonged laboratory cultivation or by rhizosphere passage. All the isolated variants were more motile than the wild-type strain and appear to contain mutations in the gacA and/or gacS gene. By disrupting these genes and complementation analysis, we have observed that the Gac system regulates swimming motility by a repression pathway. Variants isolated after selection by prolonged cultivation formed a single population with a swimming motility that was equal to the motility of gac mutants, being 150% more motile than the wild type. The motility phenotype of these variants was complemented by the cloned gac genes. Variants isolated after rhizosphere selection belonged to two different populations: one identical to the population isolated after prolonged cultivation and the other comprising variants that besides a gac mutation harbored additional mutations conferring higher motility. Our results show that gac mutations are selected both in the stationary phase and during rhizosphere colonization. The enhanced motility phenotype is in turn selected during rhizosphere colonization. Several of these highly motile variants were more competitive than the wild-type strain, displacing it from the root tip within 2 weeks.     

Suppressor mutation in Pseudomonas aeruginosa.

Suppressor mutations were identified in Pseudomonas aeruginosa, and a comparison was made with Escherichia coli suppressor systems. A suppressor-sensitive (sus) derivative of a plasmid, RP4 trp, and several Sus mutants of IncP1 plasmid-specific phages, were isolated by using E. coli. Plasmid RP4 trp (sus) was transferred to P. aeruginosa strains carrying trp markers which did not complement RP4 trp(sus), and Trp+ variants were selected. Some, but not all such revertants, could propagate PRD1 Sus phages, and these mutants were found to be supressor positive. Plating efficiencies of various Sus phages on these strains were compared with on E. coli strains carrying known suppressor genes. The results suggested that the Pseudomonas suppressors were probably amber suppressors. In iddition, some Sus phages (PRD1sus-55, PRD1sus-56) were obtained which, although apparently of the amber type for E. coli, were able to propagate equally well on sup+ or sup strains of P. aeruginosa. On the other hand, several mutants of phage PRR1 which were suppressed in E. coli were not suppressed by the P. aeruginosa suppressor. Suppressor-sensitive mutants were also isolated with P. aeruginosa bacteriophages E79 and D3.     

Rhizosphere Selection of Highly Motile Phenotypic Variants of Pseudomonas fluorescens with Enhanced Competitive Colonization Ability

Phenotypic variants of Pseudomonas fluorescens F113 showing a translucent and diffuse colony morphology show enhanced colonization of the alfalfa rhizosphere. We have previously shown that in the biocontrol agent P. fluorescens F113, phenotypic variation is mediated by the activity of two site-specific recombinases, Sss and XerD. By overexpressing the genes encoding either of the recombinases, we have now generated a large number of variants (mutants) after selection either by prolonged laboratory cultivation or by rhizosphere passage. All the isolated variants were more motile than the wild-type strain and appear to contain mutations in the gacA and/or gacS gene. By disrupting these genes and complementation analysis, we have observed that the Gac system regulates swimming motility by a repression pathway. Variants isolated after selection by prolonged cultivation formed a single population with a swimming motility that was equal to the motility of gac mutants, being 150% more motile than the wild type. The motility phenotype of these variants was complemented by the cloned gac genes. Variants isolated after rhizosphere selection belonged to two different populations: one identical to the population isolated after prolonged cultivation and the other comprising variants that besides a gac mutation harbored additional mutations conferring higher motility. Our results show that gac mutations are selected both in the stationary phase and during rhizosphere colonization. The enhanced motility phenotype is in turn selected during rhizosphere colonization. Several of these highly motile variants were more competitive than the wild-type strain, displacing it from the root tip within 2 weeks.     

Bacteriophage and phenotypic variation in Pseudomonas aeruginosa biofilm development

A current question in biofilm research is whether biofilm-specific genetic processes can lead to differentiation in physiology and function among biofilm cells. In Pseudomonas aeruginosa, phenotypic variants which exhibit a small-colony phenotype on agar media and a markedly accelerated pattern of biofilm development compared to that of the parental strain are often isolated from biofilms. We grew P. aeruginosa biofilms in glass flow cell reactors and observed that the emergence of small-colony variants (SCVs) in the effluent runoff from the biofilms correlated with the emergence of plaque-forming Pf1-like filamentous phage (designated Pf4) from the biofilm. Because several recent studies have shown that bacteriophage genes are among the most highly upregulated groups of genes during biofilm development, we investigated whether Pf4 plays a role in SCV formation during P. aeruginosa biofilm development. We carried out immunoelectron microscopy using anti-Pf4 antibodies and observed that SCV cells, but not parental-type cells, exhibited high densities of Pf4 filaments on the cell surface and that these filaments were often tightly interwoven into complex latticeworks surrounding the cells. Moreover, infection of P. aeruginosa planktonic cultures with Pf4 caused the emergence of SCVs within the culture. These SCVs exhibited enhanced attachment, accelerated biofilm development, and large regions of dead and lysed cells inside microcolonies in a manner identical to that of SCVs obtained from biofilms. We concluded that Pf4 can mediate phenotypic variation in P. aeruginosa biofilms. We also performed partial sequencing and analysis of the Pf4 replicative form and identified a number of open reading frames not previously recognized in the genome of P. aeruginosa, including a putative postsegregational killing operon.     

In situ growth rates and biofilm development of Pseudomonas aeruginosa populations in chronic lung infections

The growth dynamics of bacterial pathogens within infected hosts are a fundamental but poorly understood feature of most infections. We have focused on the in situ distribution and growth characteristics of two prevailing and transmissible Pseudomonas aeruginosa clones that have caused chronic lung infections in cystic fibrosis (CF) patients for more than 20 years. We used fluorescence in situ hybridization (FISH) directly on sputum specimens to examine the spatial distribution of the infecting P. aeruginosa cells. Mucoid variants were present in sputum as cell clusters surrounded by an extracellular matrix, whereas nonmucoid variants were present mainly as dispersed cells. To obtain estimates of the growth rates of P. aeruginosa in CF lungs, we used quantitative FISH to indirectly measure growth rates of bacteria in sputum samples (reflecting the in vivo lung conditions). The concentration of rRNA in bacteria isolated from sputa was measured and correlated with the rRNA contents of the same bacteria growing in vitro at defined rates. The results showed that most cells were actively growing with doubling times of between 100 and 200 min, with some growing even faster. Only a small stationary-phase subpopulation seemed to be present in sputa. This was found for both mucoid and nonmucoid variants despite their different organizations in sputum. The results suggest that the bacterial population may be confronted with selection forces that favor optimized growth activities. This scenario constitutes a new perspective on the adaptation and evolution of P. aeruginosa during chronic infections in CF patients in particular and on long-term infections in general.     

Nah plasmids of the IncP-9 group in natural Pseudomonas strains

Polymerase chain reaction studies showed that naphthalene-utilizing bacteria isolated from various localities of Belarus most often contained Nah plasmids of the P-9 incompatibility group and plasmids of indefinite systematics. The conventional incompatibility test and restriction enzyme analysis revealed three new IncP-9 subgroups: ζ, η, and IncP-9-like. In addition to the known nucleotide sequences of nahG and nahAc, two novel nahG variants were revealed by a restriction enzyme analysis of amplification products. An amplified rDNA restriction enzyme analysis (ARDRA) demonstrated that the native hosts of IncP-9 Nah plasmids were fluorescent bacteria of the genus Pseudomonas (P. fluorescens, P. putida, P. aeruginosa, and Pseudomonas sp.) and nonfluorescent bacteria of indefinite systematics.     

A major Pseudomonas aeruginosa clone common to patients and aquatic habitats.

The genomic relatedness of 573 Pseudomonas aeruginosa strains from environmental and clinical habitats was examined by digesting the genome with the rare-cutting enzyme SpeI. Thirty-nine strains were collected from environmental habitats mainly of aquatic origin, like rivers, lakes, or sanitary facilities. Four hundred fifty strains were collected from 76 patients with cystic fibrosis (CF) treated at four different centers, and 25 additional clinical isolates were collected from patients suffering from other diseases. Twenty-nine P. aeruginosa isolates were collected from the environment of one CF clinic. Thirty strains from culture collections were of environmental and clinic origin. A common macrorestriction fingerprint pattern was found in 13 of 46 CF patients, 5 of 29 environmental isolates from the same hospital, in a single ear infection isolate from another hospital, and 8 of 38 isolates from aquatic habitats about 300 km away from the CF clinic. The data indicate that closely related variants of one major clone (called clone C) persisted in various spatially and temporally separated habitats. Southern analysis of the clonal variants with six gene probes and two probes for genes coding for rRNA revealed almost the same hybridization patterns. With the exception of the phenotypically rapidly evolving CF isolates, the close relatedness of the strains of the clone was also shown by their identical responses in pyocin typing, phage typing, and serotyping. Besides clone C, three other P. aeruginosa clones were isolated from more than one clinical or environmental source.     

Activation of the lac genes of Tn951 by insertion sequences from Pseudomonas cepacia.

We have identified three transposable gene-activating elements from Pseudomonas cepacia on the basis of their abilities to increase expression of the lac genes of the broad-host-range plasmid pGC91.14 (pRP1::Tn951). When introduced into auxotrophic derivatives of P. cepacia 249 (ATCC 17616), this plasmid failed to confer the ability to utilize lactose. The lac genes of Tn951 were poorly expressed in P. cepacia and were not induced by isopropyl-beta-D-thiogalactopyranoside. Lac+ variants of the pGC91.14-containing strains which formed beta-galactosidase at high constitutive levels as a consequence of transposition of insertion sequences from the P. cepacia genome to sites upstream of the lacZ gene of Tn951 were isolated. Certain of the elements also increased gene expression in other bacteria. For example, IS407 strongly activated the lacZ gene of Tn951 in Pseudomonas aeruginosa and Escherichia coli, and IS406 (but not IS407) did so in Zymomonas mobilis. The results indicate that IS elements from P. cepacia have potential for turning on the expression of foreign genes in a variety of gram-negative bacteria.     

Terminal truncations in amp C beta-lactamase from a clinical isolate of Pseudomonas aeruginosa.

AmpC beta-lactamases from strains of Pseudomonas aeruginosa have previously been shown to be heterogeneous with respect to their isoelectric point (pI). In order to elucidate the origin of this heterogeneity enzymes were isolated from a clinical isolate of a multiresistant P. aeruginosa strain and biochemically characterized. The purification was accomplished in four chromatographic steps comprising dye-affinity, size-exclusion, hydrophobic interaction chromatography, and chromatofocusing; this resulted in five forms with pI values of 9.1, 8.7, 8.3, 8.2, and 7.6. When analysed by SDS/PAGE and agarose IEF each separated beta-lactamase appeared to be both size- and charge-homogeneous. The specific activities of the variants were very similar. MS of each isolated beta-lactamase form showed minor differences in molecular mass (range 40.0-40.8 kDa). MS of the beta-lactamase with a pI of 8.2 demonstrated the presence of two subforms. The N-terminal sequences of three of the beta-lactamases were identical to the published sequence [Lodge, J.M. , Minchin, S.D., Piddock, L.J.V. & Busby, J.W. (1990) Biochem. J. 272, 627-631], while two variants were truncated by two amino-acid residues, one of which was acidic. The previously published sequence contains an alanine as the ultimate residue, but two of the beta-lactamases showed a substitution of Ala371 for arginine, whereas in the remaining forms C-terminal truncations by one and three residues were found. Our results indicate that the P. aeruginosa strain does not harbour multiple copies of the ampC gene, but rather that the five beta-lactamase isoforms are products of a single structural gene. The combinations of the identified N- and/or C-terminal truncations explained the multiple pI values of the beta-lactamase isoforms.     

SP-A1 and SP-A2 variants differentially enhance association of Pseudomonas aeruginosa with rat alveolar macrophages

Chronic airway inflammation caused by Pseudomonas aeruginosa is an important feature of cystic fibrosis (CF). Surfactant protein A (SP-A) enhances phagocytosis of P. aeruginosa. Two genes, SP-A1 and SP-A2, encode human SP-A. We hypothesized that genetically determined differences in the activity of SP-A1 and SP-A2 gene products exist. To test this, we studied association of a nonmucoid P. aeruginosa strain (ATCC 39018) with rat alveolar macrophages in the presence or absence of insect cell-expressed human SP-A variants. We used two trios, each consisting of SP-A1, SP-A2, and their coexpressed SP-A1/SP-A2 variants. We tested the 6A(2) and 6A(4) alleles (for SP-A1), the 1A(0) and 1A alleles (for SP-A2), and their respective coexpressed SP-A1/SP-A2 gene products. After incubation of alveolar macrophages with P. aeruginosa in the presence of the SP-A variants at 37 degrees C for 1 h, the cell association of bacteria was assessed by light microscopy analysis. We found 1) depending on SP-A concentration and variant, SP-A2 variants significantly increased the cell association more than the SP-A1 variants (the phagocytic index for SP-A1 was approximately 52-95% of the SP-A2 activity); 2) coexpressed variants at certain concentrations were more active than single gene products; and 3) the phagocytic index for SP-A variants was approximately 18-41% of the human SP-A from bronchoalveolar lavage. We conclude that human SP-A variants in vitro enhance association of P. aeruginosa with rat alveolar macrophages differentially and in a concentration-dependent manner, with SP-A2 variants having a higher activity compared with SP-A1 variants.     

Cystic fibrosis-niche adaptation of Pseudomonas aeruginosa reduces virulence in multiple infection hosts

The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host.     

Antibiotic sensitivity of different antigenic variants of Pseudomonas aeruginosa isolated in a hospital

The serological typing of 96% of P. aeruginosa strains isolated in hospitals has been carried out by means of agglutinating sera, and the tendency towards the prevalence of the strains of group V (25.3%) and serovar O11 (18.1%) has been revealed. The determination of the antibiotic sensitivity of different variants has shown that the strains of groups I, V and serovar O11 possess pronounced resistance to a wide spectrum of antibiotics, their resistance to gentamycin and polymixin being higher than in the strains of other serotypes. The determination of antibiotic resistance can serve only as an additional test in the serological typing of P. aeruginosa.     

Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa

The slime polysaccharides produced by Pseudomonas aeruginosa isolated from a variety of human infections were investigated. Slime production in culture seemed optimal when adequate amounts of carbohydrate were present and under conditions of either high osmotic pressure or inadequate protein supply. The polysaccharides produced by the organisms were similar to each other, to the slime of Azotobacter vinelandii, and to seaweed alginic acids. They were composed of beta-1,4-linked d-mannuronic acid residues and variable amounts of its 5-epimer l-guluronic acid. All bacterial polymers contained o-acetyl groups which are absent in the alginates. The polysaccharides differed considerably in the ratio of mannuronic to guluronic acid content and in the number of o-acetyl groups. The particular composition of the slime was not found to be characteristic for the disease process from which the mucoid variants of P. aeruginosa were obtained.     

Diversity of genetic systems responsible for biodegradation of naphthalene in Pseudomonas fluorescens strains

The genetic systems that are responsible for naphthalene catabolism were analyzed in 18 naphthalene-degrading Pseudomonas fluorescens strains isolated from oil-contaminated soils in different regions of Russia. It was found that thirteen strains contain plasmids, from 20 to 120 kb in size, at least five of which are conjugative and bear the catabolic genes responsible for the complete utilization of naphthalene and salicylate. Five plasmids belong to the P-7 incompatibility group, and two plasmids belong to the P-9 incompatibility group. The naphthalene biodegradation genes of P. fluorescens are highly homologous to each other. The study revealed a new group of the nahAc genes and two new variants of the nahG gene. The suggestion is made that the key genes of naphthalene biodegradation, nahAc and nahG, evolve independently and occur in P. fluorescens strains in different combinations.     

Mutability in Pseudomonas viridiflava as a programmed balance between antibiotic resistance and pathogenicity

Mutable bacterial cells are defective in their DNA repair system and often have a phenotype different from that of their wild‐type counterparts. In human bacterial pathogens, the mutable and hypermutable phenotypes are often associated with general antibiotic resistance. Here, we quantified the occurrence of mutable cells in Pseudomonas viridiflava, a phytopathogenic bacterium in the P. syringae complex with a broad host range and capacity to live as a saprophyte. Two phenotypic variants (transparent and mucoid) were produced by this bacterium. The transparent variant had a mutator phenotype, showed general antibiotic resistance and could not induce disease on the plant species tested (bean). In contrast, the mucoid variant did not display mutability or resistance to antibiotics and was capable of inducing disease on bean. Both the transparent and mucoid variants were less fit when grown in vitro, whereas, in planta, both of the variants and wild‐types attained similar population densities. Given the importance of the methyl‐directed mismatch repair system (MMR) in the occurrence of mutable and hypermutable cells in human bacterial pathogens, we investigated whether mutations in mut genes were associated with mutator transparent cells in P. viridiflava. Our results showed no mutations in MMR genes in any of the P. viridiflava cells tested. Here, we report that a high mutation rate and antibiotic resistance are inversely correlated with pathogenicity in P. viridiflava, but are not associated with mutations in MMR. In addition, P. viridiflava variants differ from variants produced by other phytopathogenic bacteria in the absence of reversion to the wild‐type phenotype.     

Isolation of bacteriophages specific to a fish pathogen, Pseudomonas plecoglossicida, as a candidate for disease control

Two types of bacteriophage specific to Pseudomonas plecoglossicida, the causative agent of bacterial hemorrhagic ascites disease in cultured ayu fish (Plecoglossus altivelis), were isolated from diseased ayu and the rearing pond water. One type of phage, which formed small plaques, was tentatively classified as a member of the family Myoviridae, and the other type, which formed large plaques, was classified as a member of the family Podoviridae. All 27 strains of P. plecoglossicida examined, which were isolated from diseased ayu from geographically different areas in 1991 to 1999, exhibited quite similar sensitivities to either type of phage. One strain of P. plecoglossicida was highly virulent for ayu, and the 50% lethal dose (LD(50)) when intramuscular injection was used was 10(1.2) CFU fish(-1); in contrast, phage-resistant variants of this organism were less virulent (LD(50), >10(4) CFU fish(-1)). Oral administration of phage-impregnated feed to ayu resulted in protection against experimental infection with P. plecoglossicida. After oral administration of P. plecoglossicida cells of this bacterium were always detected in the kidneys of control fish that did not receive the phage treatment, while the cells quickly disappeared from the phage-treated fish. Bacterial growth in freshwater was lower in the presence of phage, and the number of phage PFU increased rapidly. These results suggest that it may be possible to use phage to control the disease caused by P. plecoglossicida.