A conservative amino acid mutation in the chromosome-encoded dihydrofolate reductase confers trimethoprim resistance in Streptococcus pneumoniae.

Multidrug-resistant Streptococcus pneumoniae strains have emerged over the past decade at an alarming rate. The molecular mechanism of trimethoprim resistance was investigated in 5 pneumococcal strains isolated in the Washington, DC, area from patients with invasive infections. Cloning and sequencing of the trimethoprim resistance determinant from these pneumococci indicated that an altered chromosome-encoded dihydrofolate reductase (DHFR) was responsible for the observed resistance. Comparison of DHFR sequences from pneumococcal strains with various susceptibilities to trimethoprim, together with site-directed mutagenesis, revealed that substitution of isoleucine-100 with a leucine residue resulted in trimethoprim resistance. Hydrogen bonding between the carbonyl oxygen of isoleucine-100 and the 4-amino group of trimethoprim is proposed to play a critical role in the inhibition of DHFR by trimethoprim. This enzyme-substrate model should facilitate the design of new antibacterial agents with improved activity against S. pneumoniae.

Identification of a gene involved in assembly of Actinomyces naeslundii T14V type 2 fimbriae.

The nucleotide sequence of the Actinomyces naeslundii T14V type 2 fimbrial structural subunit gene, fimA, and the 3' flanking DNA region was determined. The fimA gene encoded a 535-amino-acid precursor subunit protein (FimA) which included both N-terminal leader and C-terminal cell wall sorting sequences. A second gene, designated orf365, that encoded a 365-amino-acid protein which contained a putative transmembrane segment was identified immediately 3' to fimA. Mutants in which either fimA or orf365 was replaced with a kanamycin resistance gene did not participate in type 2 fimbriae-mediated coaggregation with Streptococcus oralis 34. Type 2 fimbrial antigen was not detected in cell extracts of the fimA mutant by Western blotting with anti-A. naeslundii type 2 fimbrial antibody, but the subunit protein was detected in extracts of the orf365 mutant. The subunit protein detected in this mutant also was immunostained by an antibody raised against a synthetic peptide representing the C-terminal 20 amino acid residues of the predicted FimA. The antipeptide antibody reacted with FimA isolated from the recombinant Escherichia coli clone containing fimA but did not react with purified type 2 fimbriae in extracts of the wild-type strain. These results indicate that synthesis of type 2 fimbriae in A. naeslundii T14V may involve posttranslational cleavage of both the N-terminal and C-terminal peptides of the precursor subunit and also the expression of orf365.

Decreased susceptibility to imipenem among penicillin-resistant Streptococcus pneumoniae.

We assessed the antimicrobial susceptibilities of 59 penicillin-intermediate or penicillin-resistant pneumococci. All strains were susceptible to vancomycin and rifampicin. The frequency of strains with decreased susceptibility to cefotaxime, chloramphenicol, imipenem and meropenem was 15, 31, 47 and 49% respectively. The high percentage of penicillin-intermediate or penicillin-resistant Streptococcus pneumoniae with decreased susceptibility to third-generation cephalosporins, chloramphenicol and carbapenems limits the therapeutic options for the treatment of invasive pneumococcal infections and particularly of meningitis.

Specific inhibitors of bacterial adhesion: observations from the study of gram-positive bacteria that initiate biofilm formation on the tooth surface.

Oral surfaces are bathed in secretory antibodies and other salivary macromolecules that are potential inhibitors of specific microbial adhesion. Indigenous Gram-positive bacteria that colonize teeth, including viridans streptococci and actinomyces, may avoid inhibition of adhesion by host secretory molecules through various strategies that involve the structural design and binding properties of bacterial adhesins and receptors. Further studies to define the interactions of these molecules within the host environment may suggest novel approaches for the control of oral biofilm formation.

Cloning, expression, sequence analysis, and site-directed mutagenesis of the Tn5306-encoded N5-(carboxyethyl)ornithine synthase from Lactococcus lactis K1.

The gene (ceo) encoding N5-(carboxyethyl)ornithine synthase (EC 1.5.1.24) has been isolated from the sucrose-nisin transposon Tn5306 of Lactococcus lactis K1, sequenced, and expressed at high level in Escherichia coli. The cloned enzyme has allowed the synthesis of the novel N omega-carboxypropyl amino acids N5-(1-carboxypropyl)-L-ornithine and N6-(1-carboxypropyl)-L-lysine. Comparison of the deduced amino acid sequence of N5-(1-carboxyethyl)-L-ornithine synthase (M(r) = 35,323) to the functionally analogous octopine and nopaline synthases from crown gall tumors showed surprisingly little similarity. However, N5-(1-carboxyethyl)-L-ornithine synthase and yeast saccharopine dehydrogenase exhibit homology at their N and C termini, which suggests that these two proteins constitute a distinct branch of the amino acid dehydrogenase superfamily. A centrally located 9-amino acid segment (GSGNVAQGA) in N5-(1-carboxyethyl)-L-ornithine synthase is virtually identical with a sequence present in the beta alpha beta-fold of the nucleotide binding domain of several microbial NADPH-dependent glutamate dehydrogenases. A much longer sequence of approximately 80 residues has significant similarity to alanine dehydrogenase. Substitution of arginine 15 of N5-(1-carboxyethyl)-L-ornithine synthase by lysine resulted in loss of enzyme activity.

Penicillin-resistant pneumococci from pediatric patients in the Washington, DC, area.

OBJECTIVE: To assess the prevalence and antimicrobial susceptibility of penicillin-resistant pneumococci (PRP) isolated from patients in a pediatric hospital. METHODS: All (108) isolates of Streptococcus pneumoniae recovered from usually sterile body sites between June 1, 1992, and May 31, 1993, were screened for susceptibility to penicillin by the E-test method. Minimum inhibitory concentrations of penicillin and other antibiotics were also determined by an agar dilution method for 10 PRP and 22 penicillin-susceptible strains. RESULTS: Fourteen isolates (12.9%) were PRP by the E-test; nine of these (8.3%) were intermediately resistant and five (4.6%) were highly resistant. All strains were sensitive to rifampin and vancomycin. Increased frequency of resistance to oral and parenteral cephalosporins and carbapenems was found among PRP; for most of these antibiotics, resistance exceeded 40% of the PRP. In addition, 20% of the PRP were resistant to macrolides and all penicillin-susceptible and PRP were resistant to a combination of trimethoprim and sulfamethoxazole. CONCLUSIONS: The decreased susceptibility to oral and parenteral cephalosporins, macrolides, a combination of trimethoprim and sulfamethoxazole, and carbapenems creates a significant problem in the treatment of pneumococcal infections in both ambulatory and hospitalized patients.

Enzymatic synthesis and characterization of N(5)-(carboxymethyl)-L-ornithine and N (6)-(carboxymethyl)-L-lysine.

This report describes the enzyme-catalyzed synthesis, characterization, and chromatographic separation of N(6)-(carboxymethyl)-L-lysine and N(5)-(carboxymethyl)-L-ornithine. The two N (ω) -(carboxyalkyl)amino acids are formed via a reductive condensation between glyoxylate and theε- orδ-amino groups of lysine and ornithine, respectively. Both reactions are catalyzed by the NADPH-dependent enzyme, N(5)-(carboxyethyl)ornithine synthase [EC 1.5.1.24], found in some strains of the lactic acid bacteriumLactococcus lactis subsp.lactis.

Purification and properties of fructokinase I from Lactococcus lactis. Localization of scrK on the sucrose-nisin transposon Tn5306.

Two electrophoretically distinct proteins with fructokinase (ATP:fructose-6-phosphotransferase) activity were detected in Lactococcus lactis subsp. lactis K1. Whereas fructokinase I was induced specifically by growth of the organism on sucrose, fructokinase II was derepressed during growth on ribose, galactose, maltose, and lactulose. Fructokinase I was purified about 1000-fold to electrophoretic homogeneity (specific activity 112 units/mg). The amino acid composition, N-terminal sequence, nucleoside triphosphate, and metal requirement(s) of the enzyme are reported. Ultracentrifugal analysis showed that the enzyme was primarily dimeric with subunits of 33.5 kDa (+/- 5%). When completely reduced, fructokinase I migrated as a single protein (Mr = 32,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in the absence of reducing agent two polypeptides (apparent Mr = 29,000 and 31,000) were detected. Isoelectric focusing also revealed two polypeptides (pI 5.6 and 5.8), and both species catalyzed the phosphorylation of fructose and mannose. Hybridization studies showed that: (i) a sucrose-negative mutant lacking the fructokinase I gene (scrK) retained fructokinase II activity and (ii) scrK is closely linked to scrA and scrB which encode Enzyme IIScr and sucrose-6-phosphate hydrolase, respectively. In L. lactis K1, these genes and the N5-(1-carboxyethyl)-L-ornithine synthase gene (ceo) are encoded on the sucrose-nisin transposon Tn5306 in the order ceo-scrKAB.

Transposon-encoded sucrose metabolism in Lactococcus lactis. Purification of sucrose-6-phosphate hydrolase and genetic linkage to N5-(L-1-carboxyethyl)-L-ornithine synthase in strain K1.

Sucrose-6-phosphate hydrolase from Lactococcus lactis subsp. lactis K1-23 (formerly Streptococcus lactis K1-23) has been purified 600-fold to electrophoretic homogeneity. Purification of the enzyme was achieved by DEAE-Sephacel, phosphocellulose P-11, and gel exclusion (Ultrogel AcA 54) chromatography. The purified enzyme (specific activity 31 units/mg) catalyzed the hydrolysis of both 6-O-phosphoryl-alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside (sucrose 6-phosphate) and sucrose (Km = 0.1 and 100 mM, respectively). Ultracentrifugal analysis of sucrose-6-phosphate hydrolase indicated an Mr = 52,200. The purified enzyme migrated as a single protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000). However, four distinct polypeptides were detected by analytical electrofocusing, and all four species hydrolyzed sucrose and sucrose 6-phosphate. The amino acid composition of sucrose-6-phosphate hydrolase, and the sequence of the first 12 amino acids from the NH2 terminus, have been determined. Hybridization studies with oligonucleotide probes show that the genes for sucrose-6-phosphate hydrolase (scrB), Enzyme IIScr of the phosphoenolypyruvate-dependent sucrose:phosphotransferase system (scrA), and N5-(carboxyethyl)ornithine synthase (ceo) are encoded by the same approximately 20-kilobase EcoRI fragment. This fragment is part of a large transposon Tn5306 that also encodes the nisin precursor gene, spaN, and IS904. In L. lactis ATCC 11454, spaN, IS904, scrA, and scrB (but not ceo) are encoded on a related transposon, Tn5307.

Simultaneous loss of N5-(carboxyethyl)ornithine synthase, nisin production, and sucrose-fermenting ability by Lactococcus lactis K1.

A spontaneous derivative of Lactococcus lactis subsp. lactis K1 (formerly Streptococcus lactis K1) lacking N5-(carboxyethyl)ornithine synthase (EC 1.5.1.24) was isolated. This mutant had also lost the abilities to ferment sucrose and to produce the antibiotic nisin. Hybridization studies indicate that these linked traits are encoded on the chromosome of L. lactis K1 and that they may be located on a conjugative transposon.

Use of lytic bacteriophage for Actinomyces viscosus T14V as a probe for cell surface components mediating intergeneric coaggregation.

A lytic bacteriophage for Actinomyces viscosus T14V (the reference strain for actinomyces coaggregation group A) was isolated from raw sewage. This phage, designated BF307, also lysed the T14V-derived nonfimbriated mutant PK455-2 as well as A. viscosus MG-1 and T14AV but not the other serotype 2 or serotype 1 strains of this species that were tested or any of nine Actinomyces naeslundii isolates. Phages BF307 belonged to Bradley morphological group C and was similar in appearance to the A. viscosus MG-1 phages Av-1 and Av-3, which do not productively infect A. viscosus T14V. A. viscosus MG-1 mutants selected for resistance to phage BF307, Av-3, or CT7 (a human dental plaque isolate with the same host range as BF307) were coresistant to the other two phages but sensitive to Av-1. These results indicate that the receptors on A. viscosus MG-1 for phages BF307, Av-3, and CT7 are identical or share a common precursor and that the receptor for phage Av-1 is distinct. Comparison of the genomes of BF307, Av-3, and CT7 revealed that their DNAs were similar in size but distinguishable by restriction analysis. Two altered coaggregation phenotypes were identified among the phage BF307-resistant mutants of strains MG-1, T14V, T14AV, and PK455-2. Class I mutants had lost the ability to interact with coaggregation group 1 streptococci, and class II mutants did not coaggregate with either group 1 or group 2 streptococci. These results are consistent with the proposal that the phage BF307 receptor on these A. viscosus strains is related to one of the structures that mediates coaggregation with oral streptococci. A model to delineate the various coaggregation mediators on the surface of actinomyces coaggregation group A cells is presented, and the use of these phages to probe surface components of human oral actinomyces strains is discussed.

Molecular basis of bacterial adhesion in the oral cavity.

The two varieties of fimbriae identified on oral strains of actinomyces have distinct functional properties. The type 1 fimbriae of Actinomyces viscosus T14V mediate attachment to saliva-treated hydroxyapatite. Type 2 fimbriae--on A. viscosus and the only fimbriae detected on A. naeslundii WVU45--are associated with lectin activity. Interaction of these fimbriae with complementary receptors initiates bacterial attachment to Streptococcus sanguis 34 and sialidase-treated epithelial cells and the killing of actinomyces by polymorphonuclear leukocytes (PMNs). Galactose, N-acetylgalactosamine (GalNAc), and related oligosaccharides inhibit these processes, and mutants lacking type 2 fimbriae do not participate in them. The actinomyces lectin is similar to lectins from Ricinus communis and Bauhinia purpurea that agglutinate certain strains of oral streptococci, block attachment of actinomyces to epithelial cells, and inhibit killing of actinomyces by PMNs. The S. sanguis receptor for the actinomyces lectin comprises repeating hexasaccharide units with GalNAc termini. Used as probes, the peanut agglutinin, with specificity for Gal(beta-3)GalNAc, and the lectin from B. purpurea detect a 160-kilodalton (kdal) band in SDS-PAGE-separated epithelial cell extracts and a 100-kdal band in PMN extracts. These may be receptors for type 2 fimbriae. A. viscosus genes encoding subunits of types 1 and 2 fimbriae have been cloned in Escherichia coli; the type 1 subunit is 65 kdal and the type 2 subunit is 59 kdal. Submandibular immunization of mice with a mixture of type 1 and type 2 fimbriae evokes the production of IgA and IgG antibodies in serum and saliva that inhibit in vitro adsorption of A. viscosus to SHA. These antibodies may modulate colonization of teeth by this organism.

Expression of Actinomyces viscosus antigens in Escherichia coli: cloning of a structural gene (fimA) for type 2 fimbriae.

A cosmid gene library of Actinomyces viscosus T14V was prepared in Escherichia coli to examine the expression of A. viscosus antigens and to gain insight into the structure of A. viscosus type 1 and type 2 fimbriae. Out of this library of 550 clones, 28 reacted in a colony immunoassay with antibodies against A. viscosus cells. The proteins responsible for these reactions were identified in three clones. Clones AV1209 and AV2009 displayed nonfimbrial antigens with subunits of 40 and 58 kilodaltons, respectively. Clone AV1402 showed a 59-kilodalton protein that reacted with monospecific antibody against type 2 fimbriae and that comigrated with a subunit of type 2 fimbriae during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that AV1402 expresses a gene (fimA) for a subunit of A. viscosus type 2 fimbriae.

More sensitive test agar for detection of dextranase-producing oral streptococci and identification of two glucan synthesis-defective dextranase mutants of Streptococcus mutans 6715.

A more sensitive test agar was developed to detect oral microbes with relatively low dextranase activity and to identify dextranase-negative mutants. Several oral streptococci that had previously been scored as dextranase negative readily decolorized the new, blue dextran-containing medium. To assess whether dextranase plays a role in glucan synthesis by oral streptococci, various glucan synthesis-defective mutants were tested for dextranase activity on the new medium. Mutants 4 and 27, which do not cause smooth-surface caries and which synthesize more soluble glucan than their parent, Streptococcus mutans 6715-13, were markedly deficient in these dextranase activity tests.

Characterization of glucosyltransferase-deficient, plasmid-containing mutants of Streptococcus mutans LM-7.

The possibility that glucosyltransferase (GT)-mediated insoluble-glucan synthesis from sucrose is controlled by the 3-megadalton plasmid pAM7 in Streptococcus mutans LM-7 has been examined. A low-sucrose agar medium was developed to readily detect and quantitate presumptive GT-negative mutants. Such mutants were isolated from Todd-Hewitt broth cultures grown either with or without sodium dodecyl sulfate (10 microgram/ml) or acriflavine (0.5 microgram/ml) at frequencies ranging from about 0.01 to 1%. Independently isolated mutants had the following characteristics: (i) cells were virtually devoid of cell-associated GT and did not aggregate upon addition of sucrose; (ii) cell-free culture fluids synthesized 10X less insoluble glucan than those of the parent; and (iii) cultures grown with sucrose did not form adherent deposits on the wall of the culture tube, as is typical of S. mutans. Both parent and mutants formed relatively little soluble glucan in 1-h assays. Three independently isolated mutants and the parent were found to contain similar amounts of plasmid DNA. Analysis by sucrose density gradient centrifugation and agarose gel electrophoresis did not reveal a size difference between the plasmids from parent and mutants. These results show that (i) S. mutans LM-7 generates GT-deficient mutants at relatively high frequency that still contain a 3-megadalton plasmid; (ii) both cell-associated and extracellular GT levels are depressed in the mutants, which suggests that these activities are directly or indirectly controlled by the same gene or by genes that segregate as a unit.

Adherence of Veillonella species mediated by extracellular glucosyltransferase from Streptococcus salivarius.

The effect of extracellular products from Streptococcus salivarius on sucrose-dependent adherence to smooth surfaces by other oral bacteria was studied in vitro. Strains of Streptococcus mitis, Streptococcus pyogenes, and Veillonella parvula without innate ability to adhere to a steel wire were able to do so when incubated with sucrose and cell-free culture fluid from S. salivarius strains 9759, 25975, CNII, and MEPI. These culture fluids synthesized more adherent material and water-insoluble glucan than those from Streptococcus mutans C67-1 and seven other S. salivarius strains. Among the S. salivarius strains, glucosyltransferase (GT; dextransucrase, EC 2.4.1.5) activity varied more than 100-fold. Cells of Veillonella and S. mitis S3 that had been incubated in culture fluids from S. salivarius 25975 and 9759, respectively, and then washed adhered upon subsequent incubation with sucrose. This was due to adsorbed GT because (i) the adherence was sensitive to dextranase; (ii) it was observed only with the high-GT culture fluids; (iii) it was dependent on sucrose; and (iv) the washed Veillonella cells synthesized glucan, but not fructan, from sucrose. These results suggest that sucrose-dependent adherence of bacteria without such innate ability can be mediated by (i) entrapment in insoluble glucan synthesized by S. salivarius culture fluids, and (ii) prior adsorption of GT from S. salivarius culture fluids. The possibility that GT formed by high-yield strains of S. salivarius is distributed through the mouth by the action of salivary flow and contributes to sucrose-dependent adherence and plaque formation is considered.

Analysis of the exudate produced by Streptococcus mutans SL-1 colonies of sucrose-containing agar media.

The watery exudate produced by Streptococcus mutans SL-1 colonies on sucrose-containing agar media was found to contain about 7% (wt/vol) of a water-soluble, branched dextran, 4% sucrose, and smaller (less than 1%) amounts of fructose, Folin-phenol-positive material, and lactic acid.

Occurrence and distribution of sucrose-metabolizing enzymes in oral streptococci.

Specific growth rates, growth yields, and the level and cellular distribution of three sucrose-metabolizing enzyme activities were determined for seven oral streptococci (Streptococcus mutans strains E49, BHT, 10449, SL-1, and LM-7, S. sanguis 10558, and S. salivarius 25975). Cultures were grown in a fermentor at pH 6 with either 20 mM glucose or 10 mM sucrose. Generation times varied between 21 and 70 min. Whereas some strains grew 10 to 50% more slowly with sucrose than with glucose, others did not. Growth was always logarithmic, and the growth yields were similar. Glcosyl transferase (EC 2.4.1.5) was largely extracellular; in sucrose cultures it was appreciably lower, but no major shift to a cell-associated form was found. In glucose cultures, the activity varied between 4 and 140 IU per 6-liter culture. The glucan formed was mostly or exclusively water insoluble. Glcosyl transferase was stimulated weakly (60% or less) by various dextrans. Fructosyl transferase (EC 2.4.1.10) was primarily extracellular (except in glucose cultures of S. salivarius) and varied between 0 and 337 IU/culture. In S. salivarius, the extracellular fructosyl transferase was induced by sucrose. In all S. Mutans cultures, the total fructosyl transferase activity was lower after growth with sucrose. All strains had extra- and intracellular invertase (EC 3.2.1.26) activity. Total levels varied between 210 and 3,500 IU/culture. Less extracellular activity was present in sucrose cultures. Only S. salivarius had appreciable activity in the cellular particulate fraction. Invertase activity was significantly higher than the combined glucosyl and fructosyl transferase activities in all cultures.