furans and trisubstituted pyrroles that possess an aryl sulfanyl or alkyl sulfanyl substituent at C3 has been developed.

furans and trisubstituted pyrroles that possess an aryl sulfanyl or alkyl sulfanyl substituent at C3 has been developed.

A series of homo-binuclear lanthanide complexes have been prepared from DO3A derived systems containing sevencoordinate binding domains linked via an aromatic. The luminescence properties of xylyl bridged complexes show that the lanthanide ions behave as isolated centres, while the combination of the lanthanide contraction and the lipophilicity of the linker group limits inner-sphere hydration around the metal centres for later lanthanides such as ytterbium.

Phospholipid molecular species present in 32 isolates of Clostridium difficile were examined by fast atom bombardment-mass spectrometry in negative-ion mode. This revealed major anions consistent with the expected presence of the following phosphatidylglycerol (PG) analogs: PG(31:2), PG(32:1), PG(33:2), PG(33:1), PG(34:2), and PG(34:1). The major phospholipid molecular species are distinct from those of other bacterial groups examined.Clostridium difficile is the major cause in humans of pseudomembranous colitis and is also implicated in both antibiotic-associated colitis and antibiotic-associated diarrhea (11). Nocosomial acquisition has led to numerous outbreaks of infection in hospitals and in the community (2). The emerging importance of this pathogenic species has stimulated interest in its biochemistry.Analysis of phospholipid constituents of Clostridium spp. by chromatographic approaches has revealed that Clostridium acetobutylicum typically elaborates phosphatidylethanolamine and then phosphatidylglycerol (PG), diphosphatidylglycerol, and unknown phospholipids as its major phospholipids (9). Minor amounts of other phospholipids such as phosphatidic acid and glycerol acetal derivatives of phosphatidylmethylethanolamine plasmalogens have also been described. The three major phospholipids (see above) have also been described in Clostridium butyricum. The latter species also produces phosphatidylmethylethanolamine derivatives. Phosphatidylethanolamine derivatives were not found in Clostridium perfringens (10). In the case of C. difficile, the phospholipid classes present have not been studied.Cellular fatty acid composition of several Clostridium species has been investigated previously (8) by gas chromatographic analysis of carboxylate methyl esters. However, analyses of fatty acids generally have used whole-cell extracts (3) so that fatty acid composition of phospholipid constituents alone is unknown in Clostridium spp.The aim of this study was to obtain detailed information on major phospholipid molecular species of C. difficile.Thirty-two isolates from 11 outbreaks were the gift of E. Kaczmarski, Central Public Health Laboratory, Manchester, United Kingdom. Isolates were grown on 5% horse bloodfastidious anaerobe agar (Lab-M, Bury, United Kingdom) incubated in an anaerobic cabinet (Don Whitley Scientific) at 36ЊC for 48 h. Cells were harvested and washed with phosphate-buffered saline (5), and then suspensions were centrifuged at 3,000 ϫ g for 20 min so that a pellet of cells might be obtained. Cell pellets were frozen and then lyophilized (Modulyo; Edwards High Vacuum), prior to extraction. Extraction and analysis of phospholipids by conventional fast atom bombardment-mass spectrometry (FAB-MS) (7) were performed according to our published methods (4, 5). Because of the high polarity of phospholipids, lipid extraction was performed with methanol-chloroform (2:1 [vol/vol]) rather than with methanol-chloroform (1:2 [vol/vol]), which has commonly been employed for total lipid extraction (3). This results i...

M . A BD I , D . D R UC KE R , V . B O OT E, M . K OR A CH I A N D E . T H EA KE R . 1999. The aim of this study was to obtain detailed information on phospholipids (PL) of the medically important Candida species and to determine their possible chemotaxonomic significance. Lipids were extracted from 22 strains representing 8 Candida species and their PL molecular species distributions were determined by Fast Atom Bombardment Mass Spectroscopy (FAB MS) in negative ion mode. Fifteen major lower mass peaks (m/z 221 to 289) were attributable to the expected presence of carboxylate anions and 24 major higher mass peaks (m/z 557 to 837) were attributable to phospholipid anions. Major carboxylate peaks were of the following m/z and identities : 253, C16:1 ; 255, C16:0 ; 277, C18:3 ; 279, C18:2 ; 281, C18:1 ; and 283, C18:0. The most abundant peaks consistent with the presence of phospholipid molecular species anions include those of m/z 673, 743, 833, 834 and 836 tentatively identified as phosphatidic acid (PA) (34 : 1), phosphatidylglycerol (PG) (34 : 3), phosphtidylinositol (PI) (34 : 2) and two unknown molecular species. This profile is diagnostic for the genus Candida. Quantitative differences were observed between different Candida species. Thus, polar lipid molecular species distribution in Candida spp. has chemotaxonomic significance, especially so in the case of carboxylate anions.

Porphyromonas has lipids containing hydroxy acids and C 16:0 and iso-C 15:0 major monocarboxylic acids among others. Nothing is known of its individual phospholipid molecular species. The aim of this study was to determine molecular weights and putative identities of individual phospholipid molecular species extracted from Porphyromonas gingivalis (seven strains), P. asaccharolytica (one strain) and P. endodontalis (two strains). Cultures on Blood-Fastidious Anaerobe Agar were harvested, washed and freeze-dried. Phospholipids were extracted and separated by fast atom bombardment mass spectrometry (FAB MS) in negative-ion mode. Phospholipid classes were also separated by thin layer chromatography (TLC). The major anions in the range m/z 209±299 were consistent with the presence of the C 13 : 0 , C 15 : 0 , C 16 : 0 and C 18 : 3 mono-carboxylate anions. Major polar lipid anion peaks in the range m/z 618±961 were consistent with the presence of molecular species of phosphatidylethanolamine, phosphatidylglycerol and with unidenti®ed lipid analogues. Porphyromonas gingivalis differed from comparison strains of other species by having major anions with m/z 932, 946 and 960. Unusually, a feline strain of P. gingivalis had a major peak of m/z 736. Selected anions were studied by tandem FAB MS which revealed that peaks with m/z 653 and 946 did not correspond to commonly occurring classes of polar lipids. They were however, glycerophosphates. It is concluded that the polar lipid analogue pro®les obtained with Porphyromonas are quite different from those of the genera Prevotella and Bacteroides but reveal heterogeneity within P. gingivalis.

Fast atom bombardment-mass spectrometry (FAB-MS) was used to analyse lipid extracts of bacteria to assess its usefulness for analysing anionic phospholipids of potential chemotaxonomic value. The following micro-organisms were tested: Acinetobacter calcoaceticus, Acinetobacter sp., Citrobacter freundii, Enterobacter cloacae (2 strains), Escherichia coli (3 strains), Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Plesiomonas shigelloides, Proteus mirabilis (3 strains), Serratia liquefaciens and Serratia marcescens. Negative-ion spectra provide data for twenty-seven major carboxylate anions (m/z 209-325) and for thirty-seven major phospholipid anions (m/z 645-774). Generally, the largest carboxylate peaks were due to 16:1, 16:0, cyc17 and 18:1 while the largest phospholipid anion peaks were due to PE(32:1), PE(33:1), PE(34:1), PE(34:2), PG(30:2), PG(31:2), PG(32:2), PG(34:1) and PS(33:0). However, quantitative differences were observed. For example, Acinetobacter lacked PE (33:1) but had exceptionally high peaks at m/z 748, PS(33:0), and m/z 281, octadecanoate. Unknown 'carboxylate' peaks were detected at m/z 254, 256, 261, 268, 282 and 301. In some cases, unknown peaks appeared to constitute possible homologous series being separated by delta m/z of 14(identical to methylene). For chemotaxonomic purposes, the complexity of the data required numerical analysis. Using the Pearson coefficient of linear correlation, as a measure of association, it was possible to compare all strains analysed. Typical results for strain comparisons were as follows: Ent. cloacae vs Ent. cloacae, r = 0.90 (Ent. cloacae vs Ac. calcoaceticus, r = 0.46). Thus FAB-MS represents an excellent means of obtaining large quantities of data on polar lipids of a range of bacterial isolates, which may be suitable for chemotaxonomic purposes.

Extracted phospholipids of Escherichia coli, Proteus mirabilis and Enterobacter cloacae were examined by fast atom bombardment-mass spectrometry which yielded major peaks between m/z 225 and 761. The result of extracting freeze-dried or 'wet' cells showed that freeze-drying may be omitted although weighing of dried cells offers a useful means of standardizing the extraction procedure. Anaerobic growth quantitatively altered the chemical finger-print as a result of increase in ratio of saturated: unsaturated carboxylic acids. Growth temperature also affected profiles over the temperature range 24-45 degrees C. A less drastic influence on mass spectra was culture age, over the range 16-48 h. Comparison of spectra was possible with Pearson's coefficient of linear correlation which yielded the following values: wet and lyophilized cells, r = 0.97; aerobic and anaerobic growth, r = 0.82; 24 degrees C and 45 degrees C, r = 0.76; 16 h and 48 h, r = 0.95. These results show that although quantitative differences do occur between spectra for the same organism prepared in different ways, they are less than interspecies variation, e.g. with E. coli and P. mirabilis, r = 0.46. Any differences which are due preparation method can be overcome by standardization of technique.