Pseudomonas aeruginosa antigens as potential vaccines

Pseudomonas aeruginosa is one of the most important opportunistic bacterial pathogens in humans and animals. This organism is ubiquitous and has high intrinsic resistance to antibiotics due to the low permeability of the outer membrane and the presence of numerous multiple drug efflux pumps. Various cell-associated and secreted antigens of P. aeruginosa have been the subject of vaccine development. Among pseudomonas antigens, the mucoid substance, which is an extracellular slime consisting predominantly of alginate, was found to be heterogenous in terms of size and immunogenicity. High molecular mass alginate components (30-300 kDa) appear to contain conserved epitopes while lower molecular mass alginate components (10-30 kDa) possess conserved epitopes in addition to unique epitopes. Surface-exposed antigens including O-antigens (O-specific polysaccharide of LPS) or H-antigens (flagellar antigens) have been used for serotyping due to their highly immunogenic nature. Chemical structures of repeating units of O-specific polysaccharides have been elucidated and these data allowed the identification of 31 chemotypes of P. aeruginosa. Conserved epitopes among all serotypes of P. aeruginosa are located in the core oligosaccharide and the lipid A region of LPS and immunogens containing these epitopes induce cross-protective immunity in mice against different P. aeruginosa immunotypes. To examine the protective properties of OM proteins, a vaccine containing P. aeruginosa OM proteins of molecular masses ranging from 20 to 100 kDa has been used in pre-clinical and clinical trials. This vaccine was efficacious in animal models against P. aeruginosa challenge and induced high levels of specific antibodies in human volunteers. Plasma from human volunteers containing anti-P. aeruginosa antibodies provided passive protection and helped the recovery of 87% of patients with severe forms of P. aeruginosa infection. Vaccines prepared from P. aeruginosa ribosomes induced protective immunity in mice, but the efficacy of ribosomal vaccines in humans is not yet known. A number of recent studies indicated the potential of some P. aeruginosa antigens that deserve attention as new vaccine candidates. The outer core of LPS was implicated to be a ligand for binding of P. aeruginosa to airway and ocular epithelial cells of animals. However, heterogeneity exists in this outer core region among different serotypes. Epitopes in the inner core are highly conserved and it has been demonstrated to be surface-accessible, and not masked by O-specific polysaccharide. The use of an in vivo selection/expression technology (IVET) by a group of researchers identified a number of P. aeruginosa proteins that are expressed in vivo and essential for virulence. Two of these in vivo-expressed proteins are FptA (ferripyochelin receptor protein) and a homologue of an LPS biosynthetic enzyme. Our laboratory has identified a highly conserved protein, WbpM, and P. aeruginosa with a deficiency in this protein produces only rough LPS and became serum sensitive. Results from these studies have provided the foundation for a variety of vaccine formulations.     

Pseudomonas aeruginosa as a cause of infectious diarrhoea

Pseudomonas aeruginosa is not generally considered a cause of infectious diarrhoea. However, it was the predominant organism isolated from the faeces of 23 unrelated, hospital outpatients investigated in the course of a year for persistent (> 1 week duration) diarrhoea. To investigate the possible aetiological role of P. aeruginosa, these patient histories were reviewed and a selection of their faecal isolates were investigated in vitro (n > or = 10) and in vivo (n = 2) for virulence. The patients had a mean age of 60 years, were receiving antibiotics and/or had an underlying illness. Extensive microbiological investigations identified no other potential or recognized enteropathogen in the faeces of 20 of these patients. More than 40% of the isolates tested were able to adhere to HEp-2 cells and exhibited twitching motility (type IV pili), properties indicative of their ability to colonize the human intestine. Cytotoxic activity was demonstrated in bacterium-free cell supernatants of over 80% of isolates; supernatants of four isolates tested in infant mice were weakly enterotoxigenic. Two isolates intragastrically inoculated into clindamycin pre-treated rats established persistent infections and induced signs and symptoms of enteritis. Overall these findings suggest that P. aeruginosa can cause diarrhoea particularly in immunodeficient individuals.     

Antibiotic resistance in Pseudomonas aeruginosa: an Italian survey

In order to assess the current level of resistance to widely used antipseudomonal antibiotics in clinical isolates of Pseudomonas aeruginosa, a national survey was undertaken. Fifteen hospitals throughout Italy participated in the study. The University of Catania tested the antibiotic susceptibility of 1005 consecutive clinically significant P. aeruginosa collected from March to June 1995. Lack of susceptibility, according to NCCLS breakpoints, was at the following rates: meropenem, 9.1%; imipenem, 19.3%; ceftazidime, 13.4%; carbenicillin, 27.3%; piperacillin, 12%; ticarcillin/clavulanic acid, 22.8%; amikacin, 10.6%; and ciprofloxacin, 31.9%. About half of the isolates (44.4%) were not susceptible to at least one of the antibiotics tested.     

Penicillin-resistant mechanisms in Pseudomonas aeruginosa: binding of penicillin to Pseudomonas aeruginosa KM 338

A comparison of the binding of radioactive penicillin G to whole cells and the membrane fraction derived from Pseudomonas aeruginosa KM 338 was made. This organism has intrinsic resistance to penicillin. The binding to the membrane fraction which catalyzed peptidoglycan synthesis followed saturation type kinetics and saturation was achieved at approximately 2 nmol of penicillin G per ml, whereas binding to the whole cells was entirely of the nonsaturation type. The binding of carbenicillin to the membrane fraction was determined by competition between radioactive penicillin G and unlabeled carbenicillin for the binding sites. It was bound at the same sites in almost the same manner. When whole cells were pretreated with high concentration of unlabeled penicillin G or carbenicillin, the subsequent binding of radioactive penicillin G to the membrane fraction from carbenicillin-treated cells was entirely nonspecific, but with penicillin G-pretreated cells it was still specific. There was apparently specific binding of radioactive penicillin G to ethylenediaminetetraacetate-treated cells. P. aeruginosa KM 338 had an extremely low activity of beta-lactamase compared with other enzyme-producing organisms. This enzyme from P. aeruginosa KM 338 was of the cephalosporinase type. These data indicate that penicillin resistance of P. aeruginosa KM 338 may be a consequence of the development of a permeability barrier which prevents the antibiotic from reaching its sites of action in the cytoplasmic membrane.     

Metabolites from an Antarctic sponge-associated bacterium, Pseudomonas aeruginosa

In an ongoing survey of the bioactive potential of microorganisms associated with marine invertebrates, the culture media of a sponge-associated bacterial strain of Pseudomonas aeruginosa was found to contain metabolites which inhibit the growth of several Gram-positive microorganisms. A series of diketopiperazines (1-6) including a new natural product (6) and two known phenazine alkaloid antibiotics (7 and 8) were isolated from the culture broth of this bacterium.     

Imipenem resistance in Pseudomonas aeruginosa

In 1997 in western Austria, 9.9% of Pseudomonas aeruginosa strains from patients of general practitioners were resistant to imipenem as well as 18.2% of the isolates from hospitals and 20.2% of the strains at a university teaching hospital. Within the hospital the imipenem resistance varied from 9.9% among out-patients to 28.7% in isolates from intensive care units. In medical/surgical words, up to 15.1% of P. aeruginosa strains were resistant to imipenem. The incidence of imipenem-resistant P. aeruginosa strains correlates to the use of carbapenems. In June 1997, 10 consecutive isolates from 8 patients were obtained and typed using restriction fragment length polymorphism analysis (RFLP) and Pyocin typing. All 10 isolates were resistant to meropenem as well as to imipenem. The finding (by RFLP and Pyocin typing) of individual bacterial types in each isolate strongly contradicts the spread of infection by cross infection. However, all patients were proven to have been treated with imipenem during the 3 months prior to testing. In 1997, 13,880 g of imipenem were used at the university hospital in Innsbruck. The use of carbapenems appears to be the main cause for the increased incidence of imipenem-resistant P. aeruginosa strains.     

Meta-analysis of epidemiologic studies

BACKGROUND: A metaanalysis is often performed if risk assessment with available epidemiological data is not sensible. The results of these analyses serve mainly to quantify the risk of weak associations. MATERIAL AND METHODS: In this paper we describe methodologic issues of this approach with emphasis on the difference between metaanalysis from published data and from individual patient records. As an example we discuss studies on oral contraceptives and breast cancer. Limits of metaanalysis from published data are given. CONCLUSION: Although metaanalysis with individual data may be cost- and time-consuming their results are more reliable.     

Identification of infectious Pseudomonas aeruginosa strains in an occupational saturation diving environment

OBJECTIVES: Occupational saturation divers have various skin disorders, of which skin infections are the most serious and frequent. Pseudomonas aeruginosa is the microbe most often isolated from skin infections in divers. The purpose of the present work was (a) to report the occurrence of P aeruginosa in skin infections in operational saturation diving in the North Sea from 1987 to 1995; (b) to report the environmental occurrence of P aeruginosa in saturation diving systems, and finally (c) to identify possible relations between infection related to strains of P aeruginosa and environmental isolates of the microbe. RESULTS: During the period 1987-95, P aeruginosa was isolated from 257 skin infections in operational saturation divers. Most of the isolates related to infection by P aeruginosa show a unique growth inhibition pattern towards the normal skin flora, and the serotype pattern of P aeruginosa from skin infections is limited compared with similar infections in non-divers. In a mini-epidemiological study on board one diving vessel during one operational diving period, five significantly different DNA fragment profiles were found among the 12 isolates related to infection by P aeruginosa obtained from the saturation system. In two cases the infectious genotypes were detected in the fresh water for the saturation chambers weeks before the arrival of the infected diver. CONCLUSIONS: The most commonly used epidemiological marker for P aeruginosa world wide, also used in earlier studies, is serotyping, but with pulsed field gel electrophoresis (PFGE) miniepidemiology it was shown to be insufficient for epidemiological purposes in saturation environments. PFGE analyses were shown to be superior both to antibacterial factor and to serotyping in epidemiological analyses of P aeruginosa infections in saturation diving.     

Polyphosphate kinase as a nucleoside diphosphate kinase in Escherichia coli and Pseudomonas aeruginosa

Generation of a wide variety of nucleoside (and deoxynucleoside) triphosphates (NTPs) from their cognate nucleoside diphosphates (NDPs) is of critical importance in virtually every aspect of cellular life. Their function is fulfilled largely by the ubiquitous and potent nucleoside diphosphate kinase (NDK), most commonly using ATP as the donor. Considerable interest is attached to the consequence to a cell in which the NDK activity becomes deficient or over-abundant. We have discovered an additional and possibly auxiliary NDK-like activity in the capacity of polyphosphate kinase (PPK) to use inorganic polyphosphate as the donor in place of ATP, thereby converting GDP and other NDPs to NTPs. This reaction was observed with the PPK activity present in crude membrane fractions from Escherichia coli and Pseudomonas aeruginosa as well as with the purified PPK from E. coli; the activity was absent from the membrane fractions obtained from E. coli mutants lacking the ppk gene. The order of substrate specificity for PPK was: ADP > GDP > UDP, CDP; activity with ADP was 2-60 times greater than with GDP, depending on the reaction condition. Although the transfer of a phosphate from polyphosphate to GDP by PPK to produce GTP was the predominant reaction, the enzyme also transferred a pyrophosphate group to GDP to form the linear guanosine 5' tetraphosphate.     

Diagnostic bias in occupational epidemiologic studies

Neurofilament immunoreactivity was examined in spinal cords of rats and cats with antibodies to all three subunits (68 kD, 155 kD and 200 kD) and to different phosphorylation states of 200 kD. NFHP-, an antibody against non-phosphorylated 200 kD, labelled all rat neuronal perikarya but failed to label cat neurofilaments. In both species, the perikarya and processes of motoneurones were immunoreactive for all three subunits but most dorsal horn neuronal perikarya were not immunoreactive for 68 kD and 155 kD. Motoneuronal perikarya and proximal processes showed filamentous labelling for 68 kD but not for 155 kD in the rat, while in neither species did these show labelling with RT97, an antibody against a highly phosphorylated form of 200 kD; immunoreactivity for 200 kD was present in both filamentous (probably partially phosphorylated) and non-filamentous (non-phosphorylated) forms, but in dorsal horn neurones only the latter was present. Interpretations consistent with this data are: in rat and possibly also cat, motoneuronal neurofilaments consist of a 68 kD backbone with partially phosphorylated 200 kD sidearms, with both 155 kD and 200 kD (nonphosphorylated) subunits in a non-filamentous form; this neurofilament becomes more highly phosphorylated along the proximal processes. The dorsal horn neurones probably contain 200 kD in a non-filamentous form but may lack the other subunits.     

Sample size determination in epidemiologic studies

Sample size determination is an important issue in epidemiologic studies. Standard methods for determining sample size in cohort and case-control studies have generally been restricted to dichotomous disease and exposure variables and discrete confounding variables, and are based on simplifying assumptions that could often be unrealistic. Methods for sample size determination that make less restrictive and more realistic assumptions regarding the distribution of disease, exposure and confounding variables and which more closely parallel the analyses that are performed on the data, after the study has been conducted, have been developed in recent years. In this article some recent developments in the methodology for sample size determination in epidemiologic studies are reviewed.     

A TEM-derived extended-spectrum beta-lactamase in Pseudomonas aeruginosa

A clinical strain of Pseudomonas aeruginosa, PAe1100, was found to be resistant to all antipseudomonal beta-lactam antibiotics and to aminoglycosides, including gentamicin, amikacin, and isepamicin. PAe1100 produced two beta-lactamases, TEM-2 (pI 5.6) and a novel, TEM-derived extended-spectrum beta-lactamase called TEM-42 (pI 5.8), susceptible to inhibition by clavulanate, sulbactam, and tazobactam. Both enzymes, as well as the aminoglycoside resistance which resulted from AAC(3)-IIa and AAC(6')-I production, were encoded by an 18-kb nonconjugative plasmid, pLRM1, that could be transferred to Escherichia coli by transformation. The gene coding for TEM-42 had four mutations that led to as many amino acid substitutions with respect to TEM-2: Val for Ala at position 42 (Ala42), Ser for Gly238, Lys for Glu240, and Met for Thr265 (Ambler numbering). The double mutation Ser for Gly238 and Lys for Glu240, which has so far only been described in SHV-type but not TEM-type enzymes, conferred concomitant high-level resistance to cefotaxime and ceftazidime. The novel, TEM-derived extended-spectrum beta-lactamase appears to be the first of its class to be described in P. aeruginosa.     

Evaluation of protein A and protein G as indicator system in an ELISA for detecting antibodies in mink to Pseudomonas aeruginosa

A modified, indirect enzyme-linked immunosorbent assay (ELISA) was developed and applied in the detection of mink antibodies to Pseudomonas aeruginosa. In this assay, peroxidase conjugated protein A and protein G were evaluated as indicator systems for detecting antigen-antibody complexes. It was found that protein A has a strong affinity for mink immunoglobulins. In contrast, protein G showed no such affinity. The affinity of protein A for mink immunoglobulins was further demonstrated by immunoprecipitation assays.     

Experimental studies on bacterial product CANTASTIM derived from Pseudomonas aeruginosa with immunomodulatory properties. IV. Tyrosine phosphorylation as an effect of stimulation on different cell populations

The bacterial product derived from Pseudomonas aeruginosa (trade mark-CANTASTIM) proved immunomodulatory effects in different systems, both in vitro and in vivo experimental animal models, as well as in clinical trials. Among the results obtained regarding CANTASTIM, the following immunomodulatory properties could be mentioned: an increase of the activated T cell subpopulations and humoral-mediated immune processes, facilitation of phagocytic processes, stimulation of cytotoxic activity reflected in the improvement of the capacity of defense in several tumoral and infectious diseases. To better elucidate the intimate mechanisms by which CANTASTIM modulates the cellular functions on different cellular populations, we used tyrosine phosphorylation as an estimate of cell activation on peripheral blood lymphocytes (PBL) and a monocyte cell line (THP-1). In PBL, the treatment with CANTASTIM renders them more susceptible to CD3 stimulation than non-treated cells. In monocytes, CANTASTIM and two phospholipid components of CANTASTIM modulated in a different manner the cellular adhesion on fibronectin and tyrosine phosphorylation leading to the conclusion that these phospholipid components do not fully explain CANTASTIM modulatory properties on cell adhesion processes.     

Epidemiology of community-acquired Pseudomonas aeruginosa infections in children

The epidemiology of community-acquired Pseudomonas aeruginosa infections in children during a one-year period (January through December 1993) was evaluated. A total of 6,859 clinical samples, each one representing a separate individual with suspected infection, were cultured. Pseudomonas aeruginosa was isolated from 218 children with various infections occurring in the following order of frequency: chronic suppurative otitis media, 76.3%; appendicitis/peritonitis, 10.3%; osteomyelitis, 8.9%; skin or soft tissue infection, 6.3%; acute conjunctivitis, 3.0%; and urinary tract infection, 0.1%. A variety of O serogroups were identified: O1 (15.2%), O6 (14.7%), O11 (12.4%), O10 (11.5%), O3 (10.6%), O5 (5.1%), and O9 (4.6%). Other serogroups and nontypable strains were recovered at a frequency of 11.2% and 14.7%, respectively. Nontypable strains predominated in chronic otitis media (18.9%), while serogroups O1 (18.3%), O6 (17.5%), and O11 (17.5%) were recovered most frequently among the typable isolates. Susceptibility of Pseudomonas aeruginosa to antipseudomonadal agents was extremely high. The rate of susceptibility to ceftazidime was 99.6%, to azlocillin 98.6%, to piperacillin 98.2%, to aztreonam 97.3%, to gentamicin and netilmicin 97.7%, and to ciprofloxacin 99.1%. All isolates were susceptible to tobramycin, imipenem, and amikacin. The results might suggest that community-acquired Pseudomonas aeruginosa infections in children can be treated successfully with any antipseudomonadal antibiotic.