Physiological studies on the growth and utilization of sugars by Listeria species

Experiments, relevant to growth in milk, were done to delineate the aerobic and anaerobic growth of Listeria species on selected sugars in several media. All species grew on glucose aerobically, forming lactic acid and (or) acetic acid. Anaerobically, only lactic acid was formed; cell yields were 80% of those obtained aerobically. When incubated aerobically, small amounts (1.5 microns/mL) of isovaleric acid, 2-hydroxyisovaleric acid, and trace amounts of isobutyric acid were formed. These products were characteristically formed by 26 strains representing all the species of Listeria. Added leucine stimulated isovaleric acid formation. Anaerobic fermentations of glucose could be followed by 60 to 80% cell lysis; less lysis occurred in air. Anaerobically, only hexoses and pentoses supported growth; aerobically, maltose and lactose supported growth of some strains, but sucrose did not support growth of any strain tested. Listeria grayi and Listeria murrayi utilized the galactose and glucose moieties of lactose for growth; Listeria monocytogenes and Listeria innocua used only the glucose moiety. Glucosamine and N-acetylglucosamine supported aerobic and anaerobic growth as well as glucose, and their presence stimulated the utilization of lactose by "lactose-negative" strains. Analyses of cultures grown at 5 degrees C in sterile milk treated with glucose oxidase supported the conclusion that the glucose of the milk was the major, if not the limiting, substrate that supported growth.     

A new procedure for efficient recovery of DNA, RNA, and proteins from Listeria cells by rapid lysis with a recombinant bacteriophage endolysin.

A method for the rapid lysis of Listeria cells, employing a recombinant Listeria bacteriophage A118 lytic enzyme (PLY118), is described. The procedure can be used with all listerial species. It enables fast, efficient, and gentle recovery of DNA, RNA, or native cellular proteins from small-scale (2- to 5-ml) cultures. Moreover, this approach should be very useful in analytical detection and differentiation of Listeria strains when the release of native nucleic acids or proteins is required.     

INFLUENCE OF TEMPERATURE ON THE FATTY ACID PROFILE OF LISTERIA MONOCYTOGENES

Variation in the fatty acid profile of two Listeria monocytogenes strains grown at varying temperatures was determined. The fatty acid profiles varied greatly at different temperatures. General decreases in relative percentages of branched and medium chain (up to C16:0) fatty acids and variable changes in long chain fatty acids were found with increasing growth temperature. Individual fatty acid percentages between strains were variable. The relative percentages of unknown long chain fatty acids, detected in both strains at various temperatures, were greatest in Scott A (7.07%) and ATCC 19114 (13.15%) at 35C. Results demonstrated that L. monocytogenes had altered fatty acid profile in response to changes in growth temperature.     

Inhibition of Listeria monocytogenes and Listeria innocua by hexanoic and octanoic acids

Hexanoic acid and octanoic acid inhibited growth of 10 strains of Listeria monocytogenes and two strains of L. innocua at pH 5·0 and pH 5·5 and 20°C. Octanoic acid was more inhibitory than hexanoic acid and both were more inhibitory at pH 5·0 than at pH 5·5. The minimum inhibitory concentrations (MICs) were comparable with the concentrations of these acids that have been reported in Danish Blue cheese, where they were probably formed by the metabolism of Penicillium roquefortii . Thus hexanoic and octanoic acids may contribute to the inhibition of listerias in some cheeses.     

Rapid identification of Listeria species by using restriction fragment length polymorphism of PCR-amplified 23S rRNA gene fragments

A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level. Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA. S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI. This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants. All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium. The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua. Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures. Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains. The 158 single identifiable isolates were 92 L. monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L. innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain. The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species. Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology.     

Structural studies on lipoteichoic acids from four Listeria strains.

The lipoteichoic acids were isolated from phenol extracts of four Listeria strains representing serotypes 4a, 4b, 6a, and 6 to compare the differences in structure of amphiphilic polysaccharides from various serotypes of Listeria spp. The lipoteichoic acids from the four strains examined had the same structure in both hydrophilic chains and lipid portions. On the basis of the results of nuclear magnetic resonance spectroscopy and Smith degradation, the hydrophilic chains were shown to be 1,3-linked poly(glycerol phosphate) in which some of the glycerol residues had alpha-galactosyl substituents. The lipid portions were released by treatment with 46% hydrogen fluoride or 98% acetic acid. They were determined to be 3(1)-(2'-O-alpha-D-galactopyranosyl-alpha-D-glucopyranosyl)-1(3), 2-diacylglycerol and 3(1)-[6'-phosphatidyl-2'-O-(alpha-D-galactopyranosyl)-alpha- D-glucopyranosyl]-1(3),2-diacylglycerol. The degrees of glycosyl substitution and proportions of the two lipids varied to some extent among these four strains.     

Different cellular fatty acid pattern behaviours of two strains of Listeria monocytogenes Scott A and CNL 895807 under different temperature and salinity conditions

Cells of two strains of Listeria monocytogenes CNL 895807 and Scott A were grown to late exponential phase at different growth temperatures (37, 20 and 4 degrees C) with or without NaCl (7%), and their fatty acid compositions were analysed. The results showed that low thermal adaptation response of L. monocytogenes CNL was different than that of the Scott A strain, and it was based on both an increase of anteiso-branched-chain fatty acids and a significant decrease of straight-chain fatty acids. However, the main modifications observed in the Scott A strain when grown at a low temperature were a decrease of the proportion of ai17:0 and an increase of ai15:0. In hyperosmotic medium and over the entire temperature range (4 degrees C, 20 degrees C and 37 degrees C) the two L. monocytogenes strains showed a cellular fatty acid profile dominated by ai15:0. In addition, a decrease of the two major straight-chain fatty acids (14:0 and 16:0) was observed in the CNL strain. These results demonstrated that the CNL strain showed different behaviours of low thermal and salt adaptation to maintain membrane fluidity, which are based both on an increase of anteiso-branched-chain fatty acids, and a significant decrease of straight-chain fatty acids.     

DNA sequence heterogeneity in the gene encoding a 60-kilodalton extracellular protein of Listeria monocytogenes and other Listeria species.

Chromosomal DNA sequences from the 60 kilodalton protein gene of Listeria monocytogenes, amplified by the polymerase chain reaction, were used for restriction fragment length polymorphism differentiation of L. monocytogenes serotypes and other Listeria species. All 24 strains of L. monocytogenes examined produced an extracellular protein of molecular weight 60,000 (p60) as determined by Western blot analysis. Four of six other Listeria species had a protein that cross-reacted to antibodies to p60, but all differed in molecular weight, ranging from approximately 50,000 to 65,000. The gene encoding p60 was amplified from chromosomal DNA in all strains using polymerase chain reaction with a single primer pair. Restriction enzyme digestion with HindIII of the amplified product revealed a restriction pattern that was distinct between serotypes 1/2a and either 4b or 1/2b of L. monocytogenes. Of the other Listeria species, four strains that produced a cross-reacting protein likewise produced a polymerase chain reaction amplification product with the primer pair. Listeria innocua alone had a restriction pattern similar to that of Listeria monocytogenes serotype 4b and 1/2b. Genotypic heterogeneity, as revealed by DNA amplification and restriction endonuclease digestion of the p60 open reading frame, correlates with "electrophoretic type" grouping and may be related to differences in virulence mechanisms of Listeria monocytogenes and other Listeria species.     

Inhibition of enterobacteria and Listeria growth by lactic, acetic and formic acids

Minimum inhibitory concentrations (MIC) of undissociated lactic, acetic and formic acids were evaluated for 23 strains of enterobacteria and two of Listeria monocytogenes. The evaluation was performed aerobically and anaerobically in a liquid test system at pH intervals of between 4.2 and 5.4. Growth of the enterobacteria was inhibited at 2–11 mmol 1⁻¹, 0.5–14 mmol 1⁻¹ and 0.1–1.5 mmol 1⁻¹ of undissociated lactic, acetic and formic acids, respectively. The MIC value was slightly lower with anaerobic conditions compared with aerobic conditions. The influence of protons on the inhibition was observed for acetic acid at the low pH values. Undissociated lactic acid was 2 to 5 times more efficient in inhibiting L. monocytogenes than enterobacteria. Acetic acid had a similar inhibitory action on L. monocytogenes compared with enterobacteria. Inorganic acid (HCl) inhibited most enterobacteria at pH 4.0; some strains, however, were able to initiate growth to pH 3.8. The results indicate that the values of undissociated acid which occur in a silage of pH 4.1–4.5 are about 10–100 times higher than required in order to protect the forage from the growth of enterobacteria and L. monocytogenes.     

Taxonomic evaluation of amino acid metabolism inLegionella

Legionella utilizes amino acids as the sole source of carbon and energy. We have examined 62 strains of nine species to determine whether their amino acid metabolism correlated with species definition. Neither definitive species differences nor serogroup differences within the speciesLegionella pneumophila were found. Greatest metabolic activity of all strains was observed with glutamate, alanine, aspartate, and proline; anaerobically, the greatest reduction of methylene blue was obtained with glutamate, aspartate, histidine, and lysine. Within the genus, two major groups were observed: those species that showed little endogenous activity and metabolized a select group of amino acids, and those species that showed strong endogenous activity and appeared to actively metabolize virtually all the amino acids.     

Genetic differentiation by nucleic Acid homology I. Relationships among Mycoplasma species of man

Reich, Paul R. (National Institutes of Health, Bethesda, Md.), Norman L. Somerson, Carol J. Hybner, Robert M. Chanock, and Sherman M. Weissman. Genetic differentiation by nucleic acid homology. I. Relationships among Mycoplasma species of man. J. Bacteriol. 92:302-310. 1966.-Genetic relatedness among human mycoplasmas was evaluated by measuring the amount of nucleic acid hybrid retained on a membrane filter. Hybrids were formed from deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) derived from representative strains of seven serologically distinct human Mycoplasma species. The results indicate that serologically distinct human Mycoplasma species can also be distinguished by the homology techniques. Low-level cross-reactivity was observed among nucleic acids derived from the seven species. Genetic heterogeneity was demonstrated among three strains of M. salivarium and between two strains of M. orale type 2. In contrast, comparison of three strains and three passage levels of M. pneumoniae revealed them to be indistinguishable. M. pneumoniae appears to be the most distinct of all human mycoplasmas, as shown by both homology and the high buoyant density value of its DNA. Nucleic acids from mycoplasmas which had identical buoyant densities were in some cases differentiable. Mycoplasmas with different DNA buoyant densities were invariably distinguishable by the homology technique.     

Heteroduplex mobility assay for the identification of Listeria sp and Listeria monocytogenes strains: application to characterisation of strains from sludge and food samples

One hundred and ten Listeria sp. isolates from sewage sludge were identified according to phenotypic and genotypic methods. The Listeria sp. strains isolated from five types of sludge from three sewage treatment plants in Angers (France) and the surrounding area included L. monocytogenes (55.5%), L. innocua (29.1%), L. seeligeri (13.6%) and L. welshimeri (1.8%). The majority of L. monocytogenes strains belonged to serotypes 4b, 1/2b and 1/2a. Moreover, a heteroduplex mobility assay based on the 16S rRNA sequences was tested for its ability to identify the six species of the genus Listeria. This study, performed on 283 Listeria sp. strains from human, food and sewage sludge samples, showed that all the species were distinguishable from one another. L. innocua and L. seeligeri showed respectively three and two distinct banding patterns. Within L. monocytogenes, four groups (I-IV) were defined. The majority of food and environmental isolates were clustered in group I and it is noteworthy that group IV clustered epidemiologic isolates and strains belonging to serotypes 4b, 1/2a and 1/2b.     

Short-Chain Acids of Pseudomonas Species Encountered in Clinical Specimens

The short-chain acids of 36 strains of Pseudomonas grown on Trypticase soy agar were determined by gas-liquid chromatography. Distinct acid profiles were observed for each of the eight species tested. Propionic, isobutyric, and isovaleric acids were the principal acids detected in media extracts of P. maltophilia, P. cepacia, P. pseudoalcaligenes, P. diminuta, and P. vesiculare. The presence and relative amounts of the isobutyric and isovaleric acids clearly distinguished P. maltophilia, P. pseudoalcaligenes, and P. cepacia from other species. P. diminuta could be distinguished from P. vesiculare by the production of glutaric acid; P. testosteroni was the only species tested which produced relatively large amounts of phenylacetic acid.     

Assignment of Listeria grayi and Listeria murrayi to a single species, Listeria grayi, with a revised description of Listeria grayi.

The genomic relatedness between Listeria grayi and Listeria murrayi was reevaluated by using DNA-DNA hybridization, multilocus enzyme electrophoresis, and rRNA restriction fragment length polymorphism techniques. The high levels of similarity observed between the strains of these two species confirmed the data published since 1973 and indicated that they should be considered members of a single species. On grounds of priority, the species should be named L. grayi.     

Analysis of fatty acids from lipopolysaccharides of Bacteroides thetaiotaomicron and Bacteroides fragilis]

The aim of this study was to determine and to compare fatty acids occurring in lipopolysaccharides (LPS) isolated from B. thetaiotaomicron and B. fragilis strains of different origin. Lipopolysaccharides of three B. thetaiotaomicron strains and four B. fragilis strains were isolated by phenol-water extraction according to the procedure of Westphal and Jann (1965). Water-phase LPS fractions were then treated with nucleases and purified by ultracentrifugation as described by Gmeiner (1975). Fatty acid methyl esters, obtained by methanolysis of LPS, were analysed in gas-liquid chromatography combined with mass spectrometry (GLC-MS). Trimethylsilylated hydroxyl groups of fatty acid methyl esters were identified with GLC-MS using a method of selective ion monitoring (SIM). Lipopolysaccharides of B. thetaiotaomicron and B. fragilis strains contained long-chain (15-18 carbon atoms) fatty acids. The broad spectrum of simple long-chain and branched-chain fatty acids as well as 3-hydroxy fatty acids were detected. The main fatty acid of analyzed bacterial species was 3-hydroxy-hexadecanoic acid (3OH C16:0). Several 3-hydroxy fatty acids were detected in LPS of examined strains. Fatty acids occurring in LPS of B. thetaiotaomicron and B. fragilis strains appeared to be qualitatively similar. Quantitative differences in fatty acids composition of lipopolysaccharides isolated from strains of different origin were observed.     

Lipids of antibiotic-resistant and -susceptible members of the Enterobacteriaceae.

Lipids of antibiotic-resistant and related -susceptible strains of the Enterobacteriaceae were extracted with chloroform-methanol and characterized by thin-layer chromatography, densitometry, and fatty acid analysis using gas chromatography. Quantitative differences which correlated with antibiotic resistance existed among the phospholipids and fatty acids. A relatively higher concentration of a ninhydrin-positive phospholipid concentration with a lower amount of phosphatidylethanolamine was observed in antibiotic-resistant strains of serratia marcescens. Bacterial strains which harbored R-factor 222 had a higher ratio of phosphatidylglycerol to diphosphatidylglycerol than their respective parent strains while those strains which were resistant to the polymyxins had a lower ratio of these phospholipids. Differences in the relative amounts of certain unsaturated and cyclopropane fatty acids were observed between susceptible and resistant strains. Such differences, however, were dependent upon a particular genus and species.     

Absence of cecal secondary bile acids in gnotobiotic mice associated with two human intestinal bacteria with the ability to dehydroxylate bile acids in vitro.

Germ-free mice were orally inoculated with human intestinal 7alpha-dehydroxylating bacterial strains to evaluate their ability to transform bile acids in vivo. Three weeks after inoculation of the bacteria, cecal bile acids were examined. Among free-form bile acids, only beta-muricholic acid was detected in the cecal contents of gnotobiotic mice associated with Bacteroides distasonis strain K-5. No secondary bile acid was observed in the cecal contents of any of the gnotobiotic mice associated with 7alpha-dehydroxylating bacteria, Clostridium species strain TO-931 or Eubacterium species strain 36S.     

On the basic structure of poly(glycerophosphate) lipoteichoic acids

Poly(glycerophosphate) lipoteichoic acids from 24 Gram-positive bacteria of the genera Bacillus, Enterococcus, Lactobacillus, Lactococcus, Listeria, Staphylococcus, and the streptococcal pyogenic and oral group were analyzed. The 1,3-linked poly(glycerophosphate) structure was proved by analysis of glycerol and glycerophosphates after acid and alkaline hydrolysis. Using the molar ratios of glycolipid to phosphorus (A) and phosphomonoester to phosphorus after periodate oxidation followed by hydrazinolysis (B) or beta-elimination (C), we show that all lipoteichoic acids contain a single unbranched poly(glycerophosphate) chain and that the chain is uniformly phosphodiester-linked to C-6 of the nonreducing hexopyranosyl residue of the glycolipid moiety. On some chains minor phosphate-containing substituents were detected whose structure remains to be clarified. The lipoteichoic acids of enterococci and listeria strains were separated by hydrophobic interaction chromatography into glycolipid- and phosphatidylglycolipid-containing molecular species. The phosphatidylglycolipid moieties were structurally characterized after liberation from lipoteichoic acids with moist acetic acid. After periodate oxidation of lipoteichoic acids beta-elimination released both phosphatidic acid and the poly(glycerophosphate) chain. This indicates together with the sequence analysis of the released phosphatidylglycolipid that the phosphatidyl residue is located at C-6 of the reducing hexosyl residue of the glycolipid moiety and the poly(glycerophosphate) chain at C-6 of the nonreducing one. Together with earlier observations these results complete the evidence for the structural and possibly biosynthetic relationship between lipoteichoic acids and glycerophosphoglycolipids.     

Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species.

In this study, 468 Listeria strains were checked for the presence of phosphatidylinositol-specific phospholipase C (PI-PLC) activity by using a simple assay that consisted of overlaying colonies formed on agar plates with L-alpha-phosphatidylinositol as substrate. In this assay, PI-PLC-active colonies show turbid halos around the colonies as a result of the release of insoluble diacylglycerol from the substrate. This activity was detected only in the pathogenic species Listeria monocytogenes and was not present in any of the 167 strains of Listeria seeligeri, Listeria welshimeri, Listeria innocua, Listeria murrayi, and Listeria grayi tested. Hence, screening for PI-PLC activity permits discrimination between pathogenic and nonpathogenic Listeria species. In particular, the hemolytic but nonpathogenic species L. seeligeri can now be separated from the hemolytic and pathogenic species L. monocytogenes and L. ivanovii. The use of this assay will improve the specific detection and/or isolation of pathogenic Listeria species from clinical samples or food enrichment cultures.     

Esterase activities of cell-free extracts from 'wild' strains of leuconostocs and heterofermentative lactobacilli isolated from traditional Greek cheese

Cell-free extracts from strains of leuconostocs and heterofermentative lactobacilli were tested for esterase activities using synthetic substrates. Results showed that esterases from both lactic acid bacteria groups preferentially degraded short-chain fatty acids. Moreover, even though some similarities between strains of the same species were recorded, the observed differences were not strong enough to distinguish between species and therefore commend this technique as a tool for taxonomy of either leuconostocs or heterofermentative lactobacilli.