SUMMARY A collection of 267 strains, representing many of the principal biotypes among aerobic pseudomonads, has been subjected to detailed study, with particular emphasis on biochemical, physiological and nutritional characters. A total of 146 different organic compounds were tested for their ability to serve as sources of carbon and energy. Other characters that were studied included : production of extracellular hydrolases; nitrogen sources and growth factor requirements H-chemolithotrophy; denitrifying ability; pigment production; ability to accumulate poly-p-hydroxybutyrate as a cellular reserve material; biochemical mechanisms of aromatic ring cleavage; and nature of the aerobic electron transport system. The resultant data have revealed many hitherto unrecognized characters of taxonomic significance. As a consequence, it has become possible to recognize among the biotypes examined a limited number of species which can be readily and clearly distinguished from one another by multiple, unrelated phenotypic differences.

The Aerobic Pseudomonads a Taxonomic Study

Pseudomonas aeruginosa has emerged as an important pathogen during the past two decades. It causes between 10% and 20% of infections in most hospitals. Pseudomonas infection is especially prevalent among patients with burn wounds, cystic fibrosis, acute leukemia, organ transplants, and intravenous-drug addiction. P. aeruginosa is a common nosocomial contaminant, and epidemics have been traced to many items in the hospital environment. Patients who are hospitalized for extended periods are frequently colonized by this organism and are at increased risk of developing infection. The most serious infections include malignant external otitis, endophthalmitis, endocarditis, meningitis, pneumonia, and septicemia. The likelihood of recovery from pseudomonas infection is related to the severity of the patient's underlying disease process. The introduction of the antipseudomonal aminoglycosides and penicillins has improved substantially the prognosis of these infections. Ticarcillin and carbenicillin have been especially beneficial in neutropenic patients; however, prompt institution of therapy is mandatory for optimal benefit. Many new drugs with antipseudomonal activity, including penicillins, cephalosporins, and other beta-lactams, have been introduced in recent years and offer the potential for new approaches to therapy for these infections.

Infections Caused by Pseudomonas aeruginosa

Listeria monocytogenes and listeric infections.

Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species. They were initially found as exoproducts of the opportunistic pathogen Pseudomonas aeruginosa and described as a mixture of four congeners: α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate (Rha-Rha-C10-C10), α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoate (Rha-Rha-C10), as well as their mono-rhamnolipid congeners Rha-C10-C10 and Rha-C10. The development of more sensitive analytical techniques has lead to the further discovery of a wide diversity of rhamnolipid congeners and homologues (about 60) that are produced at different concentrations by various Pseudomonas species and by bacteria belonging to other families, classes, or even phyla. For example, various Burkholderia species have been shown to produce rhamnolipids that have longer alkyl chains than those produced by P. aeruginosa. In P. aeruginosa, three genes, carried on two distinct operons, code for the enzymes responsible for the final steps of rhamnolipid synthesis: one operon carries the rhlAB genes and the other rhlC. Genes highly similar to rhlA, rhlB, and rhlC have also been found in various Burkholderia species but grouped within one putative operon, and they have been shown to be required for rhamnolipid production as well. The exact physiological function of these secondary metabolites is still unclear. Most identified activities are derived from the surface activity, wetting ability, detergency, and other amphipathic-related properties of these molecules. Indeed, rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development.

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Rhamnolipids: diversity of structures, microbial origins and roles

Publisher Summary β -lactamases are the enzymes that are capable of hydrolyzing the β -lactam bond of penicillins and cephalosporins. The reaction catalyzed by β -lactamases with penicillins as substrates includes the rupture of the β -lactam bond to form the corresponding penicilloic acid. However, there is complexity in breakdown of the cephalosporins after β -lactamase action as there is no sporin and the formation of any single product—a fact that invalidates the iodometric assay of cephalosporinase for absolute measurement. Despite the wide and varying specificity, β -lactamases often require the 4-membered azetidinone ring to be condensed either with a thiazolidine (penicillins) or a dihydrothiazine nucleus (cephalosporins). The molecular basis of the resistance of Gram-negative bacteria to β -lactam antibiotics is very different from their Gram-positive counterparts. Some of the possible physiological role recognized for β -lactamases includes—namely, (1) hydrolysis of β -lactam antibiotics when required and (2) destruction of β -lactam antibiotics. The role of protecting the organisms that produce β -lactamases against the deleterious effects of penicillins and cephalosporins can often provide a major obstacle to the successful use of β -lactam antibiotics for therapy.

The β-Lactamases of Gram-Negative Bacteria and their Possible Physiological Role

Although Pseudomonas aeruginosa has been known to produce hemolysis on blood agar plates, attempts to obtain hemolysin in broth culture filtrates of this organism have been generally unsuccessful. Bullock and Hunter (1900) noted that filtrates of 3 to 4 week old broth cultures of this organism lysed suspensions of erythrocytes of several animal species, including man. This finding was confirmed by Landsteiner and Raubilschek (1908) and Fukuhara (1909). All of these authors agreed that the hemolysin could be demonstrated only in very old nutrient broth cultures and that filtrates from young, actively growing broth cultures were always devoid of hemolytic activity. The failure of P. aeruginosa to produce a soluble hemolysin in young broth cultures has been confirmed by this author. In 1936, Birch-Hirschfeld described a cellophane plate technique for the production of staphylococcal hemolysin. This technique was extensively employed by William and Harper (1947), Marks and Vaughan (1950) and Liu (1954) in their studies of the delta hemolysin of staphylococci. Recently this cellophane plate technique was applied to the production of the soluble hemolysin by P. aeruginosa with considerable success. Since several workers (Salvin and Lewis, 1946; Christie, 1948) have reported that the hemolysis by P. aeruginosa on blood agar could be used to differentiate this species from other pseudomonads, the hemolysin production with the cellophane plate technique was also applied to examine the hemolytic activities of several other pseudomonads which resemble P. aeruginosa. The present communication describes the findings of this study.

Survey of hemolysin production among species of pseudomonads.

Lecithinase production by microorganisms has been investigated in an attempt to use it in the classification of bacteria, and to associate formation with toxicity and virulence. The first record of this reaction was made when Nagler (1939) and Seifert (1939) observed, independently, the formation of an opalescence in human serum by the a toxin of Clostridium perfringens. It was soon found that human serum could be replaced with egg yolk (Macfarlane, Oakley, and Anderson, 1941), and the nature of the reaction was identified as the lecithinase activity which resulted in the liberation of phosphorus and choline (Macfarlane and Knight, 1941), with precipitation of fat which was the source of the opalescence. Other members of the genus Clostridium have since been shown to produce this enzyme. C. oedematiens (Crook, 1942; Hayward, 1943), C. bifermentans, C. sordellii (Hayward, 1943), and C. haemolyticum (Jasmin, 1947) are the well known lecithinase producers in the genus Clostridium. Some members of the genus Bacillus, such as B. cereus, B. mycoides, and B. anthracis (McGaughey and Chu, 1948; Colmer, 1948), and several different types of acid-fast bacilli (Toda and Urabe, 1936) were also reported to produce lecithinase. Most of the organisms which produced lecithinase were gram-positive, and only a few gram-negative bacteria were reported to have this activity. Lecithinase production by Vibrio comma and El Tor vibrio has been reported by Ruata and Caneva (1901), Kraaij and Wolff (1923), and Felsenfeld (1944). The lecithinase activities of the members of the genus Serratia (Monsour and Colmer, 1952) and of the pseudomonads (Klinge, 1957) have been reported recently. In view of the paucity of the literature on the lecithinase activity of gram-negative bacteria, a study was undertaken to see the extent of lecithinase activity by this group of organisms. The present communication describes these findings. MATERIALS AND METHODS

Lecithinase production by gram-negative bacteria

The hemolysin of Pseudomonas aeruginosa was found to function as a detergent in solubilizing various phosphatides. Incorporation of the hemolysin into reaction mixtures containing phosphatides and phospholipase c significantly increased the rates of enzyme activities. Stimulation of enzyme activity was most likely due to improved dispersion of the substrates.

Effect of the Hemolysin of Pseudomonas aeruginosa on Phosphatides and on Phospholipase c Activity

Production of protease by many bacteria was found to be inhibited by ammonium salts, and the enzyme production was more sensitive to the salts than was growth of the organisms. Inhibition of protease production by some pathogenic bacteria may result in the recognition of an exotoxin which otherwise would have been digested by the protease. In the case of Pseudomonas aeruginosa, qualitatively different toxicities could be demonstrated in the culture fluids, depending on the presence or absence of protease in such a fluid. The toxicity of the culture in the presence of a high titer of protease may be due primarily to the protease, whereas the toxicity exhibited in the absence of protease could be due to proteinacious exotoxin. Producers of high titers of protease tended to be less virulent in vivo than producers of low titers of the enzyme, which exert their toxicities by a separate exotoxin.

Inhibition of Protease Production of Various Bacteria by Ammonium Salts: Its Effect on Toxin Production and Virulence

Liu, Pinghui V. (University of Louisville, Louisville, Ky.). Factors that influence toxigenicity of Pseudomonas aeruginosa. J. Bacteriol. 88:1421–1427. 1964.—Hemolysin, lecithinase, and protease of Pseudomonas aeruginosa were produced with cellophane-covered plates and completely synthetic agar of simple composition containing α-alanine, aspartic acid, glutamic acid, glucose, and salts. The most critical factor in the production of these extracellular toxins appeared to be the concentration of phosphate, which had to be in the range of 0.003% for the lecithinase, 0.005 to 0.01% for the hemolysin, and 0.01 to 0.2% for the protease. α-Alanine and glutamic acid favored the production of the hemolysin and lecithinase, and aspartic acid supported the production of protease. The role of glucose in the production of these extracellular toxins appeared to be more important than just an energy source. It is likely that products of anaerobic metabolism of glucose are needed in the production of these toxins. In the absence of glucose, the production of protease was enhanced by lactic acid, pyruvic acid, or α-ketoglutaric acid. Possible implications of these findings in the pathogenesis of P. aeruginosa are discussed.

Pinghui V. Liu

Papers

Survey of hemolysin production among species of pseudomonads.

Lecithinase production by gram-negative bacteria

Effect of the Hemolysin of Pseudomonas aeruginosa on Phosphatides and on Phospholipase c Activity

Inhibition of Protease Production of Various Bacteria by Ammonium Salts: Its Effect on Toxin Production and Virulence

Factors that influence toxigenicity of pseudomonas aeruginosa