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PubMed1995

Gonococcal opacity: lectin-like interactions between Opa proteins and lipooligosaccharide.

Previous evidence from our laboratory suggested that the tight intercellular adhesions between the outer membranes of gonococci displaying the opacity colony phenotype occurred because Opa proteins expressed on one gonococcus adhered to the lipooligosaccharide (LOS) of the opposing bacterium (M.S. Blake, p. 51-66, in G. G. Jackson and H. Thomas, ed., The Pathogenesis of Bacterial Infections, 1985, and M. S. Blake and E. C. Gotschlich, p. 377-400, in M. Inouye, ed., Bacterial Outer Membranes as Model Systems, 1986). A noncompetitive inhibition assay used previously to determine the carbohydrate structures recognized by the major hepatic asialoglycoprotein receptor was modified to determine the gonococcal LOS structures that bind Opa proteins (R. T. Lee, Targeted Diagn. Ther. Ser. 4:65-84, 1991). The LOS carbohydrates used in these assays were LOS structures purified from pyocin LOS mutants of Neisseria gonorrhoeae 1291 described by K. C. Dudas and M. A. Apicella (Infect. Immun. 56:499-504, 1988) and further characterized by C. M. John et al. (J. Biol. Chem. 266:19303-19311, 1991). Purified gonococcal Opa proteins were incubated with each of the parent and mutant LOS, and the amount of binding of Opa proteins was measured by a direct enzyme-linked immunosorbent assay using the Opa-specific monoclonal antibody 4B12. The affinities of the Opa proteins for each of the LOS were determined indirectly by measuring the concentrations of Opa proteins that noncompetitively inhibited 50% of the binding of LOS-specific monoclonal antibodies. This concentration is inversely proportional to the affinity of the inhibitor (R. T. Lee, Targeted Diagn. Ther. Ser. 4:65-84, 1991). Our data suggest that the gonococcal Opa proteins tested had the highest affinity for the Gal beta 1-4GlcNAc residue present on the gonococcal lactoneoseries LOS. This affinity was comparable to that reported for the binding of the major hepatic asialoglycoprotein receptor to glycoconjugates containing terminal galactose and N-acetylgalactosamine (R. T. Lee, Targeted Diagn. Ther. Ser. 4:65-84, 1991). After sialylation of the lactoneoseries LOS, presumably on the terminal galactose residue, the interaction with the Opa proteins was ablated. Therefore, the gonococcal Opa-LOS and mammalian epithelial cell asialoglycoprotein receptor-carbohydrate interactions have quite similar specificities.

Antigens, Bacterial↗

PubMed2005

Use of phage display to identify potential Pseudomonas aeruginosa gene products relevant to early cystic fibrosis airway infections.

Pseudomonas aeruginosa airway infections are a major cause of morbidity and mortality in patients with cystic fibrosis. Treatment of established infections is difficult, even with microbiologically active agents. Thus, prevention of infection is an important goal of management. Isolates from cystic fibrosis patients appear to originate from the environment but adapt to the milieu of the airway of the cystic fibrosis patient and evolve toward a common phenotype. Identification of the antigens expressed early in infection may lead to novel targets for vaccine development. Immunogenic peptides were identified in a J404 random nonapeptide phage display library with serum from cystic fibrosis patients obtained within the first year of P. aeruginosa infection. One hundred sixty-five reactive clones were verified by plaque lift assays, and their inserts were sequenced. The sequenced nonapeptides were compared with the published sequence of strain PAO1, identifying homologies to 76 genes encoding outer membrane and secreted proteins. The majority of these were proteins involved in small-molecule transport, membrane structural proteins, and secreted factors. An in silico analysis was performed that suggested that the occurrence of multiple matches to predominantly outer membrane and secreted proteins was not attributable to random chance. Finally, gene expression array data from early isolates of P. aeruginosa from cystic fibrosis patients was compared with the results from phage display analysis. Eleven outer membrane and secreted proteins were common between the two data sets. These included genes involved in iron acquisition, antibiotic efflux, fimbrial biogenesis, and pyocin synthesis. These results demonstrate the feasibility and validity of this novel approach and suggest potential targets for future development.

Antibodies, Bacterial↗

PubMed1990

DNA sequences and characterization of four early genes of the tryptophan pathway in Pseudomonas aeruginosa.

Two pairs of related but easily distinguishable genes for the two subunits of anthranilate synthase have been identified in Pseudomonas aeruginosa. These were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance cassette, and returned to the P. aeruginosa chromosome, replacing the wild-type gene. Gene replacement implicated only one of the pairs in tryptophan biosynthesis. This report describes the cloning and sequencing of the tryptophan-related gene pair, designated trpE and trpG, and presents experiments implicating their gene products in tryptophan production. DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also includes trpD and trpC. Complementation of Trp auxotrophs by R-prime plasmids (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983) has shown that a large cluster of pyocin R2 genes is flanked at one end by trpE and the other end by trpDC; the physical map that was obtained shows the distance between trpE and trpDC to be about 25 kilobases. Our restriction map of the trpE and trpGDC regions agrees with data presented by Shinomiya et al.

Amino Acid Sequence↗

PubMed2000

RegA, iron, and growth phase regulate expression of the Pseudomonas aeruginosa tol-oprL gene cluster.

The tol-oprL region in Pseudomonas aeruginosa appears to be involved in pyocin uptake and required for cell viability. The complete nucleotide sequences of the tolQRA and oprL genes as well as the incomplete sequences of tolB and orf2 have been previously reported. In addition, the sequence of a P. aeruginosa iron-regulated gene (pig6) has been described and found to share homology with an open reading frame located upstream of the Escherichia coli tolQRA genes (U. A. Ochsner and M. L. Vasil, Proc. Natl. Acad. Sci. USA 93:4409-4414, 1996). In this study, we cloned the remainder of the P. aeruginosa tol-oprL gene cluster and determined its nucleotide sequence. This cluster was found to consist of seven genes in the order orf1 tolQ tolR tolA tolB oprL orf2. Transcriptional analysis of this gene cluster was performed by detecting the presence of mRNAs spanning adjacent genes as well as by using a promoterless lacZ reporter gene fused to each of the seven genes contained in the tol-oprL locus. The results show that there are three major transcriptional units or operons in this region, orf1-tolQRA, tolB, and oprL-orf2, in contrast to the E. coli tol-pal region, where there are only two operons, orf1-tolQRA and tolB-pal-orf2. Analysis of gene expression indicated that the tol-oprL genes of P. aeruginosa are both iron and growth phase modulated. The first operon, orf1-tolQRA, is iron regulated throughout growth, but iron-regulated expression of tolB and oprL fusions occurs only in late log phase. The expression of the three operons was significantly less repressed by iron in fur mutants than in the wild-type strain, suggesting the involvement of Fur in the iron regulation of all three operons. RegA is a positive yet nonessential regulator of tol-oprL expression.

Bacterial Outer Membrane Proteins↗

PubMed2002

Autolysis and autoaggregation in Pseudomonas aeruginosa colony morphology mutants.

Two distinctive colony morphologies were noted in a collection of Pseudomonas aeruginosa transposon insertion mutants. One set of mutants formed wrinkled colonies of autoaggregating cells. Suppressor analysis of a subset of these mutants showed that this was due to the action of the regulator WspR and linked this regulator (and the chemosensory pathway to which it belongs) to genes that encode a putative fimbrial adhesin required for biofilm formation. WspR homologs, related in part by a shared GGDEF domain, regulate cell surface factors, including aggregative fimbriae and exopolysaccharides, in diverse bacteria. The second set of distinctive insertion mutants formed colonies that lysed at their center. Strains with the most pronounced lysis overproduced the Pseudomonas quinolone signal (PQS), an extracellular signal that interacts with quorum sensing. Autolysis was suppressed by mutation of genes required for PQS biosynthesis, and in one suppressed mutant, autolysis was restored by addition of synthetic PQS. The mechanism of autolysis may involve activation of the endogenous prophage and phage-related pyocins in the genome of strain PAO1. The fact that PQS levels correlated with autolysis suggests a fine balance in natural populations of P. aeruginosa between survival of the many and persistence of the few.

Bacterial Adhesion↗

PubMed2006

Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth.

Two recently sequenced genomes of the insect-pathogenic bacterium Photorhabdus and a large Serratia entomophila plasmid, pADAP, have phage-related loci containing putative toxin effector genes, designated the "Photorhabdus virulence cassettes" (PVCs). In S. entomophila, the single plasmid PVC confers antifeeding activity on larvae of a beetle. Here, we show that recombinant Escherichia coli expressing PVC-containing cosmids from Photorhabdus has injectable insecticidal activity against larvae of the wax moth. Electron microscopy showed that the structure of the PVC products is similar to the structure of the antibacterial R-type pyocins. However, unlike these bacteriocins, the PVC products of Photorhabdus have no demonstrable antibacterial activity. Instead, injection of Photorhabdus PVC products destroys insect hemocytes, which undergo dramatic actin cytoskeleton condensation. Comparison of the genomic organizations of several PVCs showed that they have a conserved phage-like structure with a variable number of putative anti-insect effectors encoded at one end. Expression of these putative effectors directly inside cultured cells showed that they are capable of rearranging the actin cytoskeleton. Together, these data show that the PVCs are functional homologs of the S. entomophila antifeeding genes and encode physical structures that resemble bacteriocins. This raises the interesting hypothesis that the PVC products are bacteriocin-like but that they have been modified to attack eukaryotic host cells.

Animals↗

PubMed1968

Viability in fertile stocks of Pseudomonas aeruginosa.

Diluent composition, time consumed in experimental manipulations, and the presence of pyocine-like lethal agents affected the viability of sexual fertility factor-positive and fertility factor-negative auxotrophic stocks of Pseudomonas aeruginosa. In the same cultures, recovery of prototrophic revertants increased as the number of viable cells dispensed per plate was reduced. As a result, the number of revertants recovered was indirectly determined by the combined activities of the three conditions affecting viability. Possible modifications by these conditions affecting viability on the expression and interpretation of the fertility factor-positive sex factor-mediated system of genetic recombination are presented.

Bacteriocins↗

PubMed1984

Association of infection caused by Pseudomonas aeruginosa serotype O11 with intravenous abuse of pentazocine mixed with tripelennamine.

From July 1979 to June 1983, 25 of 40 intravenous drug addicts with systemic infections had Pseudomonas aeruginosa as the etiological agent; by 1982, P. aeruginosa had replaced Staphylococcus aureus as the most common pathogen. At least 21 of the 25 addicts with P. aeruginosa infection abused pentazocine mixed with tripelennamine (commonly known as T's and blues) compared with 6 of 15 addicts infected with other pathogens (P = 0.006). Of the 25 P. aeruginosa isolates, 23 were of serotype O11. Phenotypic patterns in isolates from addicts and in 22 serotype O11 control isolates from nonaddicts were determined by pyocin and electrophoretic enzyme typing, as well as by susceptibility to heavy metals and antibiotics. Of 25 isolates from addicts, 20 were identical or differed by only one marker, whereas the 22 nonaddict serotype O11 isolates were distributed among 17 distinct phenotypic patterns. We postulate that the emergence of P. aeruginosa as the major cause of deep infection in addicts is a consequence of contamination of their paraphernalia during preparation of pentazocine and tripelennamine for self-injection. The phenotypic similarity among isolates from addicts may reflect acquisition from related environmental sources and an unusual ability of certain serotype O11 strains to survive preparation of the drugs or to be invasive.

Drug Contamination↗

PubMed1993

Pseudomonas aeruginosa folliculitis acquired through use of a contaminated loofah sponge: an unrecognized potential public health problem.

Pseudomonas aeruginosa folliculitis is a well-known entity that occurs among users of closed-cycle recreational water sources such as whirlpools, swimming pools, and hot tubs. In the absence of this epidemiologic link, isolated cases are difficult to diagnose. We encountered a patient who developed P. aeruginosa folliculitis subsequent to the use of a loofah sponge grossly contaminated with the same P. aeruginosa strain (serotype 10; pyocin type 1/a 4,b) that was recovered from her skin lesions. Furthermore, we demonstrated that sterile unused loofah sponges can serve as the sole growth-promoting substrate for P. aeruginosa. To obviate the potential public health problem of contaminated loofah sponges, it is strongly recommended that manufacturers append, and consumers adhere to, instructions as to the care of loofah sponges, which includes allowing the sponge to dry after use.

Adult↗

PubMed1974

Aeruginocine typing of Pseudomonas aeruginosa.

Aeruginocine typing using eight indicator strains of Wahba on Bacto tryptone-glucose yeast extract Agar (Difco) with an incubation temperature of 32 degrees C was found to be a reproducible and easy method which could be readily adopted by a diagnostic clinical laboratory. The results of aeruginocine typing of 1500 strains of Ps. aeruginosa suggest that hospitals differ in the range and patterns of aeruginocine types of strains isolated in them. In addition to representatives of nine of the 10 pyocine types of Wahba, a large number of strains isolated in India were found to fall into other groups not described in Wahba's system. Hence, 20 new inhibition patterns (aeruginocine types) are proposed.

Bacteriocins↗

PubMed1977

Cross infection in a surgical ward caused by Pseudomonas aeruginosa with transferable resistance to gentamicin and tobramycin.

An outbreak of gentamicin- and tobramycin-resistant Pseudomonas aeruginosa infection occurred in a surgical ward over a three-month period. Resistant Ps. aeruginosa strains with the same serological, phage, and pyocin type were cultured from the urine of six patients. Identical organisms were found on urine bottles, bedpans, and the hands of attendant staff. Inadequate disinfection played a major role in cross-infection. Isolates of the epidemic strain from each of the patients and of an unrelated but similarly resistant Ps. aeruginosa from one of them could transfer resistance to a recipient strain of Ps. aeruginosa. Resistance to gentamicin, kanamycin, tobramycin, sulphonamides, and mercuric chloride was determined by R factors belonging to Pseudomonas incompatibility group P-3. Aminoglycoside resistance was due to acetylation.

Aged↗

PubMed1988

Pseudomonas aeruginosa: in vitro susceptibility to antimicrobial drugs, single and combined, with and without defibrinated human blood.

Twelve clinical isolates of Pseudomonas aeruginosa of distinct pyocin type varied in susceptibility to 14 of 17 antimicrobial drugs. The 2 x MIC concentrations of 16 antimicrobial drugs combined with 55% (v/v) of fresh, defibrinated human blood yielded additive effects. Additive effects were noted with blood plus the MIC concentrations of all drugs tested except cefoperazone, gentamicin, and netilmicin. Blood combined with subinhibitory (1/2 MIC) concentrations of aztreonam, ceftazidime, ciprofloxacin, fleroxacin, imipenem, and tobramycin, respectively, yielded additive effects; indifferent effects were observed with the remaining 10 blood plus 1/2 MIC drug combinations. The following drug combinations additively augmented the antibacterial activity of 65% (v/v) of human blood against two selected isolates of P. aeruginosa: tobramycin (1 microgram/ml) plus the MIC or 2 x MIC concentrations of azlocillin, aztreonam, ceftazidime, ciprofloxacin, imipenem, norfloxacin, ofloxacin, piperacillin, and ticarcillin, respectively. Imipenem (8 micrograms/ml) combined with ceftazidime, cefoperazone, and piperacillin, but not aztreonam, enhanced the bactericidal activity of human blood. Rifampin (2 micrograms/ml) plus tobramycin (0.5-1 microgram/ml) combined with 8 or 16 micrograms/ml of azlocillin, aztreonam, cefoperazone, ceftazidime, imipenem, and piperacillin, respectively, enhanced blood-mediated killing of three representative multiple-drug-resistant P. aeruginosa isolates. Additional effective triple-drug combinations with human blood were rifampin + tobramycin + polymyxin B, rifampin + ciprofloxacin + imipenem, and rifampin + amikacin + imipenem. Ciprofloxacin (2 micrograms/ml) was the most potent intraphagocytic bactericidal drug of 16 tested agents (greater than or equal to 2 x MBC concentrations) against P. aeruginosa control strain ATCC 27853.

Anti-Bacterial Agents↗

PubMed1977

Persistence of a specific pseudomonas infection in a large general hospital.

Of at least 37 strains of Pseudomonas aeruginosa distinguishable by their pyocine production, Type 10 is noteworthy for its relative rarity and its virulence to man. Opportunistic infections by this organism occurring in a single hospital over 8 years are described, and its ecology discussed with reference to its possible control.

Cross Infection↗

PubMed1979

Differences in attachment antigens of gonococci in reinfection.

On the basis of bacterial typing, pyocin typing, and antibiotic sensitivity tests, two consorts appeared to have been reinfected 34 and 41 days later, respectively, with the same gonococcal strain, suggesting a failure in these patients to develop effective immunity to reinfection. However, these tests do not measure antigens which mediate attachment, a function which may correlate with virulence. When the above infecting strains were retested in an inhibition-of-attachment assay using rabbit gonococcal antisera, the antigens mediating attachment were found to be different. Homologous antisera inhibited attachment of the homologous strain at a high titer. Absorbing the antisera with the initial infecting strain did not remove any of the blocking activity of the antisera raised to the reinfecting strai, but the latter strain did share some attachment antigens in common with the initial infecting strain. Homologous antisera also bound preferentially to pili purified from the homologous strains.

Antigen-Antibody Reactions↗

PubMed1990

Complex typing of Pseudomonas aeruginosa strains isolated in an intensive care unit [a note].

A total of 2637 Gram-negative facultatively pathogenic bacteria were isolated in the László Central Hospital for Infectious Diseases during a 5-year-period from clinical samples of patients of the Respiratory Intensive Care Unit; 28 further strains were cultured from hospital personnel and fomites. Pseudomonas aeruginosa, Klebsiella and Acinetobacter spp. were most frequently isolated. Complex typing (determination of O serogroup, phage pattern, pyocin type, antibiogram and plasmid pattern) of P. aeruginosa strains showed the predominance of serogroup O11 (62%), but the isolates differed from each other by other characteristics. Conjugation experiments showed no common resistance plasmids in the tested population.

Adolescent↗

PubMedDate not supplied

[Introduction of King A and King B media for the diagnosis of Pseudomonas aeruginosa at the Teaching Pediatric Hospital of Centro Habana].

The use of King A and King B media for the faster diagnosis of Pseudomonas aeruginosa is introduced in the Microbiology Laboratory of Centro Habana Pediatric Teaching Hospital. The appropriate use of these media allows to classify as Pseudomonas aeruginosa 77.2% of the Pseudomonas sp. strains studied, which represent 95.2% of the total of strains identified as Pseudomonas aeruginosa, according to Hugh and Gilardi conventional technique. There are no significant differences in the implementation of the two procedures (X2: 0.5; p less than 0.05). The demonstrations of pyocines with King A medium is significantly superior to the method for extraction and solubility of the pigment in chloroform (X2: 15.05; p less than 0.01).

Bacterial Typing Techniques↗

PubMed1986

[Hospital infection due to Pseudomonas aeruginosa in resuscitation departments].

The bacteriological survey of resuscitation and intensive care units has revealed the presence of P. aeruginosa strains in the microflora in 69.4% of cases. The circulation of 1-2 P. aeruginosa strains, identical in their serovar and pyocin type, is indicative of the presence of hospital infection and, therefore, the endogenous character of the contamination of patients. P. aeruginosa hospital strains are the main causative agents of infectious complications in the patients treated in resuscitation units.

Bronchi↗

PubMed1987

Lipopolysaccharide structure and serum sensitivity of non-serogroupable Neisseria meningitidis.

Bactericidal activities of normal human serum for non-serogroupable strains of Neisseria meningitidis were determined. In similar experiments with isolates of Neisseria gonorrhoeae from localized infections, strains with group I lipopolysaccharide (LPS) were uniformly serum resistant and those with group II were serum sensitive. We found no similar association between serum sensitivity of the meningococcal strains and their lipopolysaccharide groups determined by the same pyocin typing system used to classify the gonococcal isolates. Immune mouse sera raised against non-serogroupable meningococci of either LPS group I or II were bactericidal for non-serogroupable strains of the same LPS group and also cross-reactive for strains of the opposite group. They were not bactericidal for the majority (13/17) of the serogroupable strains tested. These findings suggest there are antigens, in addition to the LPS and capsules, that elicit some of the "natural" bactericidal antibodies to pathogenic meningococci.

Animals↗