The esterification of fatty acids by Staphylococcus aureus fatty acid modifying enzyme (FAME) and its inhibition by glycerides.

Fifty-five randomly selected Staphylococcus aureus strains were examined for fatty acid modifying enzyme (FAME) production. Of these, 20.4% did not elaborate the enzyme. Amongst the remaining strains, the lowest level produced in culture was 0.1 unit/10(9) cocci and the maximum was 2.01 U/10(9) cocci; the median level was 0.4 U/10(9) cocci. In a series of straight-chain saturated fatty acids with 11-24 carbons, all could be esterified by FAME. However, those with 15-19 carbons were generally better substrates than the others. For a particular chain length, the unsaturated forms were better substrates than the saturated form. Triglycerides with unsaturated fatty acid side chains were potent inhibitors of FAME. Diglycerides were almost as active as triglycerides, but monoglycerides were much less inhibitory. FAME was purified by gel filtration followed by hydrophobic interaction chromatography on hexyl agarose. FAME and lipase may have a role in determining the survival of S. aureus in lesions.     

Differentiation of Serratia marcescens and Serratia liquefaciens by tests for lipase and phospholipase production.

The production of lipase and phospolipase by certain members of the Enterobacteriaceae was examined by thin-layer chromatography of resting-cell suspensions incubated with triolein or lecithin. Most strains of Serratia marcescens produced both enzymes while most strains of Serratia liquefaciens exhibited strong lipase but only a minor phospholipase activity. Enterobacter spp. (25 strains), Klebsiella pneumoniae (20 strains), Escherichia coli (15 strains), Citrobacter freundii (7 strains) and Proteus spp. (20 strains) lacked both types of enzymic activity except for the following: three strains of Enterobacter cloacae, two of Proteus mirabilis and three of Proteus vulgaris possessed slight lipase activity; about one-half of the Enterobacter aerogenes and Enterobacter hafniae strains examined produced slight phospholipase activity. It is suggested that tests for lipase and phospholipase should be used in conjunction with those for DNAase production and sugar fermentation for the differentiation of S. marcescens and S. liquefaciens.     

Hydrolytic enzymes of Moraxella bovis.

Certain strains of Moraxella bovis produce tissue-damaging enzymes which may initiate or potentiate infectious bovine keratoconjunctivitis. Thirteen reference strains of this species were characterized physiologically and screened for production of various enzymes by some conventional biochemical tests and the API ZYM system (Analytab Products, Plainview, N.Y.). All 13 strains were hemolytic. All hydrolyzed Tween 80 and Tween 85 and displayed C4 esterase, C8 esterase-lipase, and C14 lipase activities. All produced phosphoamidase and phosphatase. All were able to hydrolyze casein and gelatin. All produced leucine and valine aminopeptidases and fibrinolysin. Twelve produced hyaluronidase or were agarolytic. Three hydrolyzed chondroitin sulfate. Nine strains autoagglutinated. Five produced catalase, and two produced cystine aminopeptidase.     

Identification and partial characterisation of an extracellular activator of fatty acid modifying enzyme (FAME) expression in Staphylococcus epidermidis.

Fatty acid modifying enzyme (FAME) is an extracellular enzyme that inactivates bactericidal fatty acids by esterifying them to cholesterol. Inactivation of these fatty acids may allow Staphylococcus epidermidis to live for long periods of time on the skin. This study describes the identification and partial characterisation of an extracellular activator of FAME production. Addition of FAME-free concentrated culture filtrate (activator) to S. epidermidis cultures (OD600 = 0.05) caused a 3-5-fold increase in FAME activity. Addition of the activator did not increase the amount of exopolysaccharide produced by S. epidermidis. The mol. wt of this activator was <3000 kDa and it was quite resistant to boiling. Treatment of the activator with proteinase K did not destroy its ability to induce FAME expression. Addition of S. aureus activator to S. epidermidis cultures also increased FAME expression. However, when S. epidermidis activator was added to S. aureus cultures no increase or inhibition in FAME production was observed.     

Role of lipase in <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> complex (Bcc) invasion of lung epithelial cells

The Burkholderia cepacia complex (Bcc) is a group of ten closely related species associated with life-threatening infection in cystic fibrosis (CF). These bacteria are highly antibiotic resistant, with some strains transmissible, and in a subgroup of patients, they can cause a rapid and fatal necrotising pneumonia. The Bcc organisms produce a range of exoproducts with virulence potential, including exopolysaccharide, proteases and lipases. Many members of the Bcc are also capable of epithelial cell invasion, although the mechanism(s) involved are poorly understood. This study investigates a role for Bcc lipase in epithelial cell invasion by Bcc strains. Lipase activity was measured in eight species of the Bcc. Strains that produced high levels of lipase were predominantly from the B. multivorans and B. cenocepacia species. Pre-treatment of two epithelial cell lines with Bcc lipase significantly increased invasion by two B. multivorans strains and one B. cenocepacia strain and did not affect either plasma membrane or tight junction integrity. Inhibition of Bcc lipase production by the lipase inhibitor Orlistat significantly decreased invasion by both B. multivorans and B. cenocepacia strains in a concentration-dependent manner. This study demonstrates the extent of lipase production across the Bcc and establishes a potential role for lipase in Bcc epithelial cell invasion.     

Immunoglobulin A1 protease production by Haemophilus influenzae and Streptococcus pneumoniae.

Bacterial strains of Haemophilus species and Streptococcus pneumoniae were examined for synthesis of the enzyme immunoglobulin A1 (IgA1) protease. Of 36 H. influenzae strains examined, 35 produced IgA1 protease; strains included all six capsular types, unencapsulated variants of types b and d, and untypable H. influenzae. Eight Haemophilus strains (non-H. influenzae) were studied, and two produced IgA1 protease. All 10 strains of S. pneumoniae produced IgA1 protease; these strains included 9 different capsular polysaccharide types and 1 untypable strain. Both IgA1 proteases cleaved myeloma IgA1 and secretory IgA but not myeloma IgA2, IgM, or IgG as determined by immunoelectrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both enzymes cleaved IgA1 myeloma sera, but not IgA2, into two fragments. The apparent molecular weight of the cleaved fragments was dependent both on the apparent molecular weight of the cleaved fragments was dependent both on the specific IgA1 protease assayed and the specific IgA1 substrate utilized. It is postulated that both carbohydrate variation between the IgA1 substrates studied and the ability of S. pneumoniae glycosidases to cleave carbohydrates from glycoprotein offer an explanation for the different fragment sizes observed.     

Molecular characterization of a salt-tolerant bacterial community in the rice rhizosphere

The diversity of salt-tolerant bacteria present in the rhizosphere of Oryza sativa was investigated. Fourteen bacterial strains, isolated after enrichment in nitrogen-free, semi-solid medium and showing tolerance to 3% NaCl, were analyzed by restriction patterns produced by amplified DNA coding for 16S rDNA (ARDRA) with enzymes Sau3AI, AluI and RsaI which showed that they were represented by 4 ARDRA types. Biodiversity among the 14 strains was also analyzed by the random amplified polymorphic DNA (RAPD) technique with a 10-mer primer. Partial nucleotide sequence of 16S rDNA assigned these clusters to Serratia marcescens, Pseudomonas aeruginosa, Alcaligenes xylosoxidans and Ochrobactrum anthropi. Notably, all four bacterial species are potential human pathogens that infect immunocompromised patients.     

Fatty acid profiles in the family Leptospiraceae

Fatty acid profiles of six leptospira strains representative of genera, species, and serogroups within the family Leptospiraceae were determined by gas liquid chromatography (GLC) of fatty acid methyl ester (FAME) derivatives. The influence of methodological and biological variables on FAME profiles of the same strain was tested. FAME profiles were sharply affected by the fatty acid composition of the culture medium but not by the growth phase. Twenty-four FAME peaks were selected on the basis of their presence in repeated gas chromatographic runs of single strains. Inter-strain divergences of FAME profiles were quantified by linear regression analysis (LR). Step-wise divergences in FAME profiles were observed between strains at serogroup, species, and genus levels.     

Analysis of enzymatic activities for differentiation of two species of nutritionally variant streptococci, Streptococcus defectivus and Streptococcus adjacens.

Strains of nutritionally variant streptococci, Streptococcus defectivus (n = 10) and Streptococcus adjacens (n = 20), were studied for the production of glycosidic and proteolytic enzyme activities. S. defectivus strains produced neuraminidase and alpha-fucosidase, while S. adjacens strains produced only neuraminidase. The S. adjacens strains produced a very wide range of proteolytic activities with the ability to hydrolyze the majority of aminopeptidase substrates tested, while S. defectivus strains hydrolyzed only a minority of aminopeptidase substrates. These data provide additional phenotypic characteristics which may be useful in distinguishing between these two species and suggest that they may have different nutritional requirements.     

Differentiation between Candida dubliniensis and Candida albicans by fatty acid methyl ester analysis using gas-liquid chromatography

Candida dubliniensis is often found in mixed culture with C. albicans, but its recognition is hampered as the color of its colonies in primary culture on CHROMagar Candida varies. Furthermore, definite identification of C. dubliniensis is difficult to achieve, time-consuming, and expensive. Therefore, a method to discriminate between these two closely related yeast species by fatty acid methyl ester (FAME) analysis using gas-liquid chromatography (Sherlock Microbial Identification System [MIS]; MIDI, Inc., Newark, Del.) was developed. Although the chromatograms of these two species revealed no obvious differences when applying FAME analysis, a new library (CADLIB) was successfully created using Sherlock Library Generation Software (MIDI). The amount and frequency of FAME was analyzed using library training files (n = 10 for each species), preferentially those comprising reference strains. For testing the performance of the CADLIB, clinical isolates genetically assigned to the respective species (C. albicans, n = 32; C. dubliniensis, n = 28) were chromatographically analyzed. For each isolate tested, MIS computed a similarity index (SI) indicating a hierarchy of possible strain fits. When using the newly created library CADLIB, the SIs for C. albicans and C. dubliniensis ranged from 0.11 to 0.96 and 0.53 to 0. 93 (for all but one), respectively. Only three isolates of C. albicans (9.4%) were misidentified as C. dubliniensis, whereas all isolates of C. dubliniensis were correctly identified. Resulting differentiation accuracy was 90.6% for C. albicans and 100% for C. dubliniensis. Cluster analysis and principal component analysis of the resulting FAME profiles showed two clearly distinguishable clusters matching up with two assigned species for the strains tested. Thus, the created library proved to be well suited to discriminate between these two species.     

Ecology and nature of immunoglobulin A1 protease-producing streptococci in the human oral cavity and pharynx.

The identity and proportional distribution of immunoglobulin A1 (IgA1) protease-producing streptococci in the oral and pharyngeal microflora were studied. A collection of 459 streptococcal strains, including reference strains of Streptococcus species, and fresh isolates from human dental plaque and buccal and pharyngeal mucosa were identified by biochemical means and were examined for IgA1 protease production. IgA1 protease production was demonstrated in some, but not all, strains of Streptococcus sanguis and Streptococcus mitior and in a group of strains of uncertain taxonomic affiliation. The property was not associated with particular biotypes within the two species. Strains of S. sanguis and S. mitior isolated from Macaca fascicularis also cleaved human IgA1. A significantly different proportion of streptococcal populations in different ecosystems produced IgA1 protease. The enzyme was released by 62.7% of streptococcal isolates from buccal mucosa in contrast to only 7.8% from pharyngeal mucosa. In samples from initial and mature dental plaque 38 to 40% of streptococcal isolates produced IgA1 protease. This difference was largely a result of the frequency by which IgA1 protease activity was present in S. mitior, the predominant streptococcal species in all samples. Among otherwise identical isolates of S. mitior, 67.8% from buccal mucosa in contrast to only 5.9% from pharyngeal mucosa produced IgA1 protease. The results indicate that IgA1 protease may confer an ecological advantage to streptococci colonizing surfaces exposed to a secretory IgA-mediated selection pressure.     

Phenotypic characterization of xpr, a global regulator of extracellular virulence factors in Staphylococcus aureus.

We recently described a Tn551 insertion in the chromosome of Staphylococcus aureus S6C that resulted in drastically reduced expression of extracellular lipase (M. S. Smeltzer, S. R. Gill, and J. J. Iandolo, J. Bacteriol. 174:4000-4006, 1992). The insertion was localized to a chromosomal site (designated omega 1058) distinct from the lipase structural gene (geh) and the accessory gene regulator (agr), both of which were structurally intact in the lipase-negative (Lip-) mutants. In this report, we describe a phenotypic comparison between strains S6C, a hyperproducer of enterotoxin B; KSI9051, a derivative of S6C carrying the Tn551 insertion at omega 1058; ISP546, an 8325-4 strain that carries a Tn551 insertion in the agr locus; and ISP479C, the parent strain of ISP546 cured of the Tn551 delivery plasmid pI258repA36. Compared with their respective parent strains, ISP546 and KSI9051 produced greatly reduced amounts of lipase, alpha-toxin, delta-toxin, protease, and nuclease. KSI9051 also produced reduced amounts of staphylococcal enterotoxin B. Coagulase production was increased in ISP546 but not in KSI9051. Using a mouse model, we also demonstrated that ISP546 and KSI9051 were far less virulent than ISP479C and S6C. We have designated the genetic element defined by the Tn551 insertion at omega 1058 xpr to denote its role as a regulator of extracellular protein synthesis. We conclude that xpr and agr are similar and possibly interactive regulatory genes that play an important role in pathogenesis of staphylococcal disease.     

Prevalence of Shigella Enterotoxins 1 and 2 among Shigella Strains Isolated from Patients with Traveler's Diarrhea

Shigella spp. are known primarily as a cause of bacillary dysentery. However, in an initial phase, numerous patients exhibit watery diarrhea that may or may not be followed by dysentery. New virulence factors associated with the species of Shigella have recently been described. These are enterotoxins 1 and 2 of Shigella (ShET-1 and ShET-2, respectively). The aim of the present study was to determine the prevalence of ShET-1 and ShET-2 in species of Shigella isolated from patients with traveler's diarrhea. During the period from 1993 to 1998, stool samples from 500 travelers with diarrhea were cultured for the isolation of Shigella spp. and other enteropathogens. The detection of ShET-1 and ShET-2 was performed by a PCR technique with specific primers. Among a total of 51 strains of Shigella isolated during this period (22 S. flexneri, 26 S. sonnei, and 3 S. dysenteriae strains), at least one enterotoxin was detected in 31 (60.78%) strains; 2 (9.09%; both of which were S. flexneri strains) produced only ShET-1, while 21 (41.17%; 3 S. flexneri, 15 S. sonnei, and 3 S. dysenteriae strains) produced ShET-2. Furthermore, 8 (15.69%) of 22 S. flexneri strains presented both enterotoxins. Our results show that the prevalence of ShET-2 was high in all the Shigella species studied and confirm that ShET-1 is detected only in S. flexneri.     

Hemolysins and other characteristics that help differentiate and biotype Staphylococcus lugdunensis and Staphylococcus schleiferi.

Reference strains and clinical isolates representing the newly defined species Staphylococcus lugdunensis and Staphylococcus schleiferi were examined with the battery of tests previously recommended (G.A. Hébert, C.G. Crowder, G.A. Hancock, W.R. Jarvis, and C. Thornsberry, J. Clin. Microbiol. 26:1939-1949, 1988) for other species of coagulase-negative staphylococci (CNS). The Staph-Ident system (Analytab Products, Plainview, N.Y.) supplemented with tests for synergistic hemolysis, adherence to glass, pyroglutamyl-beta-naphthylamide hydrolysis, and susceptibility to a set of five antimicrobial disks differentiated each of these species from other species of CNS and separated strains within each species into several biotypes. Most strains (95%) of S. lugdunensis produced a delta hemolysin like that seen with nine other species of CNS. Most strains (91%) of S. schleiferi produced a beta hemolysin, which is a unique characteristic among CNS. Most (95%) of the S. schleiferi but very few (12%) of the S. lugdunensis were adherence positive. Both hemolysins and adherins are potential virulence factors among CNS. Some (29%) of the S. lugdunensis were beta-lactamase positive. The S. lugdunensis were resistant to polymyxin B and bacitracin (10 U), but the S. schleiferi were susceptible to both disks. Clinical isolates of S. lugdunensis were aligned in 18 biotypes because of eight biochemical profiles and eight physiologic subtypes; isolates of S. schleiferi were in 8 biotypes because of three biochemical profiles and subtypes. These tools for correctly identifying and then biotyping two more clinical species of CNS should enhance both epidemiologic and ecologic investigations.     

Use of PCR and sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques for differentiation of Prevotella intermedia sensu stricto and Prevotella nigrescens

Primers were designed from 16S rRNA sequences of Prevotella intermedia sensu stricto and Prevotella nigrescens and were used to discriminate these two species by PCR. The results were compared with those from the PCR technique using primers designed from arbitrarily primed PCR products by Guillot and Mouton (E. Guillot and C. Mouton, J. Clin. Microbiol. 35:1876-1882, 1997). The specificities of both assays were studied by using P. intermedia ATCC 25611, P. nigrescens ATCC 33563, 174 clinical isolates of P. intermedia sensu lato, and 59 reference strains and 58 clinical isolates of other Prevotella species and/or common oral flora. In addition, the usefulness and reliability of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the differentiation of the two species were examined by comparing the results with those from PCR assays. The controversial lipase test for distinguishing these species was also carried out. Unambiguous differentiation was made by both PCR assays, and the results matched each other. The SDS-PAGE assay was found to misidentify a few strains tested, compared with the results of PCR assays. The lipase test was positive for both species, including the reference strains of P. intermedia and P. nigrescens. We conclude that both PCR assays are simple, rapid, reliable, and specific methods which could be used in clinical studies and that the lipase test is not valuable in the differentiation. The reliable discrimination of the two species by SDS-PAGE is questionable.     

Phosphatidylinositol-specific phospholipase C, a possible virulence factor of Staphylococcus aureus.

Phosphatidylinositol-specific phospholipase C (PIPLC), an enzyme that can specifically release phosphatidylinositol-linked proteins from host cells, is one of the extracellular enzymes produced by Staphylococcus aureus. To investigate whether PIPLC might be a virulence factor, we assessed PIPLC production by S. aureus strains that had been isolated from healthy carriers and from infected patients with or without toxic shock syndrome. Although none of five vaginal isolates from healthy women was a PIPLC producer, only 10 of 32 selected pathogenic strains that caused significant infections or toxic shock syndrome elaborated PIPLC enzyme activity. Seven of 24 toxic-shock-associated strains, compared with 3 of 8 non-toxic-shock-associated strains, were positive for PIPLC. The majority of strains that produced PIPLC were negative for toxic shock syndrome toxin 1 (P less than 0.05); this association between PIPLC production and strains negative for toxic shock syndrome toxin 1 was even stronger among strains isolated only from patients with toxic shock syndrome (P less than 0.01). Among all 32 pathogenic isolates, PIPLC-producing S. aureus strains were isolated from four of four patients developing adult respiratory distress syndrome and four of five patients with disseminated intravascular coagulation, suggesting a significant association between PIPLC production and adult respiratory distress syndrome and/or disseminated intravascular coagulation (P less than 0.002). On the basis of these results, we propose that PIPLC is a virulence factor of S. aureus and is implicated in the development of adult respiratory distress syndrome and disseminated intravascular coagulation.     

Quantitative analysis and partial characterization of cytotoxin production by Salmonella strains.

The pathogenesis of the wide-spectrum human disease caused by Salmonella species is poorly understood. Cytotoxin production by other enteric pathogens has been increasingly investigated recently, and data are accumulating regarding the role of cytotoxins in enteric infections and hemolytic uremic syndrome. We studied the cytotoxic activity of 131 Salmonella strains of the major serotypes, including 94 strains of Salmonella enteritidis, 12 strains of Salmonella typhi, and 25 strains of Salmonella choleraesuis. Cytotoxicity was quantitatively determined in sonic extracts by a [3H]thymidine-labeled HeLa cell assay. All Salmonella strains examined showed some degree of cytotoxic activity. The geometric means +/- standard deviations of the amounts of cytotoxin produced (50% cytotoxic dose per milligram of bacterial protein) were 27 +/- 2 for S. typhi, 65 +/- 2 for S. enteritidis, and 117 +/- 2 for S. choleraesuis. Analysis of variance showed that the differences in cytotoxin production by the three species were significant (P less than 0.001). No significant differences were found between stool isolates and invasive strains of the same species. Neutralization studies showed that the cytotoxins produced by all Salmonella strains were immunologically distinct from Shiga toxin and the closely related Shiga-like toxins produced by Escherichia coli. DNA hybridization studies with DNA probes for Shiga-like toxins of types I and II showed no hybridization. In each species the cytotoxin was heat labile and sensitive to trypsin treatment, which indicated that its active component was probably protein in nature. Upon ultrafiltration with Amicon membranes and gel filtration chromatography, cytotoxic activity was found in the molecular weight range of 56,000 to 78,000. Our findings indicate that salmonellae produce cytotoxin(s) that may play a role in the manifestations of the various species.     

Characterisation of and polysaccharide production by amoxycillin-resistant streptococci

Small numbers of bacteria capable of growing on agar supplemented with amoxycillin 40 mg/L were isolated from the saliva of 9 out of 20 adult volunteers in a previous study. All the bacteria were identified as Streptococcus sanguis although no strains produced dextran in conventional tests. However, using a specific assay, all the antibiotic-resistant strains were found to secrete glucosyltransferases (GTF), the enzymes that synthesise these extracellular polysaccharides; the production of GTF-S, the enzyme that synthesizes dextran, was 22-43% less than that of an antibiotic-sensitive control strain. Enzyme production by both antibiotic-resistant and sensitive bacteria was markedly inhibited by dextran primer. The amoxycillin-resistant bacteria were resistant to other penicillins; their resistance to erythromycin was variable but they were uniformly sensitive to cephalothin and clindamycin. As dextran production has been proposed as a key factor in the colonisation of damaged heart valves by bacteria such as S. sanguis, these highly resistant bacteria may not pose a threat to the susceptible individual.     

Some properties of coagulase-negative deoxyribonuclease-producing strains of staphylococci from human infections

Twenty-seven coagulase-negative and deoxyribonuclease-positive staphylococci were isolated from more than 3000 specimens from human infections. The strains were tested by conventional biochemical tests and by simple agar plate assays for production of different extracellular enzymes and toxins. Three strains were lysed byS. epidermidis phages and 7 strains byS. aureus phages. All strains produced thermolabile nuclease but only 21 strains produced thermostable nuclease.The investigated strains belonged to a heterogeneous intermediate group sharing characters ofS. aureus andS. epidermidis. Tests for production of coagulase and thermostable nuclease should be used in the classification of these intermediate strains in diagnostic bacteriology.     

Production of hemolysin and other extracellular enzymes by clinical isolates of Pseudomonas pseudomallei.

One hundred clinical isolates of Pseudomonas pseudomallei from humans were tested for their ability to produce extracellular, biologically active substances which are thought to contribute to the virulence of Pseudomonas species. All isolates produced at least on extracellular enzyme; 91 strains were positive for lecithinase, lipase, and protease; but none was positive for elastase. Ninety-three strains produced a hemolysin which was detectable around the heavy growth on saline-washed sheep erythrocyte brain heart infusion agar but not demonstrable around individual colonies or in broth culture filtrate. In contrast, a hemolysin which was cytolytic around individual colonies of P. pseudomallei on the assay plate and in broth culture filtrate was exhibited by four strains. By using one of these four isolates as the test strain, the latter hemolysin was characterized further. It was heat labile, most active in an acid environment (pH 5.5), and cytolytic in broth culture filtrate for a variety of animal and human erythrocytes. Sterols, particularly cholesterol and 7-dehydrocholesterol, inhibited its hemolytic activity, but the activity was not enhanced by reducing agents or suppressed by reagents which modify sulfhydryl-activated hemolysins. A nonhemolytic mutant of the test strain of P. pseudomallei retained the extracellular enzymes of its parent, indicating that the hemolysin was not a lecithinase, lipase, or protease.