Reactive Oxygen Species Production Is a Major Factor Directing the Postantibiotic Effect of Fluoroquinolones in Streptococcus pneumoniae.

We studied the molecular mechanisms involved in the postantibiotic effect of the fluoroquinolones levofloxacin and moxifloxacin in Streptococcus pneumoniae Wild-type strain R6 had postantibiotic effects of 2.05 ± 0.10 h (mean ± standard deviation [SD]) and 3.23 ± 0.45 h at 2.5× and 10× MIC of levofloxacin, respectively. Moxifloxacin exhibited lower effects of 0.87 ± 0.1 and 2.41 ± 0.29 h at 2.5× and 10× MIC, respectively. Fluoroquinolone-induced chromosome fragmentation was measured at equivalent postantibiotic effects for levofloxacin (2.5× MIC) and moxifloxacin (10× MIC). After 2 h of drug removal, reductions were approximately 7-fold for levofloxacin and 3-fold for moxifloxacin, without further decreases at later times. Variations in reactive oxygen species production were detected after 4 to 6 h of drug withdrawals, with decreases ≥400-fold for levofloxacin and ≥800-fold for moxifloxacin at 6 h. In accordance, after 4 to 6 h of drug withdrawal, the levofloxacin-induced upregulation of the fatCDEB operon, introducing iron in the bacteria, decreased up to 2- to 3-fold, and the moxifloxacin-induced upregulation of several genes involved in the production of pyruvate was reduced 3- to 7-fold. In accordance, lower postantibiotic effects (up to 1 h) were observed in strain R6 ΔspxB, lacking the main enzyme involved in oxygen peroxide production, than in R6. Although no change in the recovery of chromosome fragmentation was observed between R6 and R6 ΔspxB, 3.5 × 103-fold lower reactive oxygen species production was observed in R6 ΔspxB, without changes after drug removal. These results show that reactive oxygen species are the main factors directing the postantibiotic effect of levofloxacin and moxifloxacin in S. pneumoniae.

Reactive Oxygen Species Contribute to the Bactericidal Effects of the Fluoroquinolone Moxifloxacin in Streptococcus pneumoniae.

We studied the transcriptomic response of Streptococcus pneumoniae to the fluoroquinolone moxifloxacin at a concentration that inhibits DNA gyrase. Treatment of the wild-type strain R6, at a concentration of 10× the MIC, triggered a response involving 132 genes after 30 min of treatment. Genes from several metabolic pathways involved in the production of pyruvate were upregulated. These included 3 glycolytic enzymes, which ultimately convert fructose 6-phosphate to pyruvate, and 2 enzymes that funnel phosphate sugars into the glycolytic pathway. In addition, acetyl coenzyme A (acetyl-CoA) carboxylase was downregulated, likely leading to an increase in acetyl-CoA. When coupled with an upregulation in formate acetyltransferase, an increase in acetyl-CoA would raise the production of pyruvate. Since pyruvate is converted by pyruvate oxidase (SpxB) into hydrogen peroxide (H2O2), an increase in pyruvate would augment intracellular H2O2. Here, we confirm a 21-fold increase in the production of H2O2 and a 55-fold increase in the amount of hydroxyl radical in cultures treated during 4 h with moxifloxacin. This increase in hydroxyl radical through the Fenton reaction would damage DNA, lipids, and proteins. These reactive oxygen species contributed to the lethality of the drug, a conclusion supported by the observed protective effects of an SpxB deletion. These results support the model whereby fluoroquinolones cause redox alterations. The transcriptional response of S. pneumoniae to moxifloxacin is compared with the response to levofloxacin, an inhibitor of topoisomerase IV. Levofloxacin triggers the transcriptional activation of iron transport genes and also enhances the Fenton reaction.

Pneumococci can persistently colonize adult patients with chronic respiratory disease.

Streptococcus pneumoniae plays an important role in causing acute exacerbations in patients with chronic respiratory disease. However, few data are available regarding pneumococcal persistence in adult patients with chronic respiratory diseases. Fifty pneumococci recovered from sputum samples (1995 to 2010) from 13 adult patients with ≥ 3 episodes of acute exacerbation or pneumonia, with the same serotype and pulsed-field gel electrophoresis (PFGE) pattern, were studied. Multilocus sequence typing (MLST) loci, penicillin-binding protein (PBP) genes (pbp2x, pbp1a, pbp2b), and the quinolone-resistant determining regions (QRDRs) of parC, parE, and gyrA were PCR amplified and sequenced. The average time between the first and last episode was 582 days (standard deviation [SD], ± 362). All but two patients received multiple courses of ß-lactam treatment, and all persistent strains were resistant to penicillin; however, the PBP sequences were stable over time apart from one variable nucleotide in pbp2x, observed among pneumococci isolated from three patients. In contrast, 7/11 patients treated with fluoroquinolones had fluoroquinolone-resistant pneumococci. In three patients, the initially fluoroquinolone-susceptible strain developed resistance after fluoroquinolone therapy, and in the remaining four patients, the persistent strain was fluoroquinolone resistant from the first episode. QRDR changes involved in fluoroquinolone resistance were frequently observed in persistent strains after fluoroquinolone treatment; however, the PBP sequences and MLST genotypes of these strains were stable over time.

Initiation of replication of plasmid pLS1. The initiator protein RepB acts on two distant DNA regions.

The broad host range streptococcal plasmid pLS1 encodes the 24.2 kDa protein RepB, which is involved in the initiation of plasmid replication by an asymmetric rolling circle. RepB was overproduced in an Escherichia coli expression system and the protein was purified and characterized. Determination of the amino-terminal sequence of RepB protein showed that translation starts from the first AUG codon, which is preceded by an atypical ribosome-binding site sequence. RepB protein has in vitro-specific endonuclease and topoisomerase-like activities on the plasmid ori(+). Footprinting experiments showed that RepB protein binds to a DNA region that includes three direct repeats of 11 base-pairs. Initiation of replication of pLS1 could start by a RepB-generated specific nick introduced on the plasmid coding strand. However, as a striking difference with other Gram-positive replicons, the nick generated by RepB lies 86 base-pairs upstream from its binding region. To explain the action of RepB at a distance, complex structures of the pLS1 ori(+) are proposed.

Proteins encoded by the DpnII restriction gene cassette. Two methylases and an endonuclease.

Proteins encoded by three genes in the DpnII restriction enzyme cassette of Streptococcus pneumoniae were purified and characterized. Large amounts of the proteins were produced by subcloning the cassette in an Escherichia coli expression system. All three proteins appear to be dimers composed of identical polypeptide subunits. One is the DpnII endonuclease, and the other two are DNA adenine methylase active at 5' GATC 3' sites. Inactivation of enzyme activity by insertions into the genes and comparison of the DNA sequence with the amino-terminal sequence of amino acid residues in the proteins demonstrated the following correspondence between genes and enzymes. The promoter-proximal gene in the operon, dpnM, encodes a 33 X 10(3) Mr polypeptide that gives rise to a potent DNA methylase. The next gene, dpnA, encodes the 31 x 10(3) Mr polypeptide of a weaker and less-specific methylase. The third gene, dpnB, encodes the 34 x 10(3) Mr polypeptide of the endonuclease. Although the endonuclease polypeptide is initiated from an ordinary ribosome-binding site, each of the methylase polypeptide begins at an atypical site with a consensus sequence entirely different from that of Shine & Dalgarno. This presumptive novel ribosome-binding site is well recognized in both S. pneumoniae and E. coli.

Molecular bases of three characteristic phenotypes of pneumococcus: optochin-sensitivity, coumarin-sensitivity, and quinolone-resistance.

Streptococcus pneumoniae is uniquely sensitive to amino alcohol antimalarials in the erythro configuration, such as optochin, quinine, and quinidine. The protein responsible for the optochin (quinine)-sensitive (Opts, Qins) phenotype of pneumococcus is the proteolipid c subunit of the FzeroF1 H(+)-ATPase. OptR/QinR isolates arose by point mutations in the atpC gene and produce different amino acid changes in one of the two transmembrane alpha-helices of the c subunit. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (Opts) and M222 (an OptR strain produced by interspecies recombination between pneumococcus and S. oralis), and S. oralis (OptR) revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin, quinine, and related compounds specifically inhibited the membrane-bound ATPase activity. Equivalent differences between Opts/Qins and OptR/QinR strains, both in growth inhibition and in membrane ATPase resistance, were found. Pneumococci also show a characteristic sensitivity to coumarin drugs, and a relatively high level of resistance to most quinolones. We have cloned and sequenced the gyrB gene, and characterized novobiocin resistant mutants. The same amino acid substitution (Ser-127 to Leu) confers novobiocin resistance on four isolates. This residue position is equivalent to Val-120 of Escherichia coli ryGB, a residue that lies inside the ATP-binding domain but is not involved in novobiocin binding in E. coli, as revealed by crystallographic data. In addition, the genes encoding the ParC and ParE subunits of topoisomerase IV, together with the region encoding amino acids 46 to 172 (residue numbers as in E. coli) of the pneumococcal ryGA subunit, were characterized in respect to fluoroquinolone resistance. The gyrA gene maps to a physical location distant from the gyrB and parEC loci on the chromosome. Ciprofloxacin-resistant (CpR) clinical isolates had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level CpR), or in both resistance-determining regions of ParC and GyrA (high-level CpR). Mutations were found in residue positions equivalent to Ser-83 and Asp-87 of the E. coli GyrA subunit. Transformation experiments demonstrated that topoisomerase IV is the primary target of ciprofloxacin, DNA gyrase being a secondary one.

Fluoroquinolones inhibit preferentially Streptococcus pneumoniae DNA topoisomerase IV than DNA gyrase native proteins.

The genes encoding the subunits of DNA topoisomerase IV (parC and parE) and DNA gyrase (gyrA and gyrB) of Streptococcus pneumoniae were cloned and overproduced in Escherichia coli by using the T7promoter-T7 RNA polymerase system. The four subunits were separately purified to near homogeneity by column chromatography. Protein purification was achieved by DEAE-sepharose, heparin-agarose, and hydroxylapatite chromatography. DNA topoisomerase IV was reconstituted when ParC and ParE were combined at a 3.8-fold excess of ParE. The reconstituted topoisomerase IV showed to generate efficient ATP-dependent DNA decatenation activity. The DNA gyrase ATP-dependent supercoiling activity was reconstituted by mixing equimolar amounts of the two gyrase subunits. The inhibitory effects of four representative fluoroquinolones on the DNA decatenation activity of topoisomerase IV and DNA supercoiling of gyrase have been examined and compared. All four compounds were more active in inhibiting topoisomerase IV than gyrase. Moreover, there was a positive correlation between the inhibitory activity against topoisomerase IV decatenation and DNA gyrase supercoiling. The classification of the four fluoroquinolones, considering their inhibitory activities in decatenation, supercoiling and growth was the following: clinafloxacin > trovafloxacin > sparfloxacin > ciprofloxacin. These results suggest these drugs primarily target topoisomerase IV of S. pneumoniae, and gyrase secondarily, in agreement with genetic data.

Ser-127-to-Leu substitution in the DNA gyrase B subunit of Streptococcus pneumoniae is implicated in novobiocin resistance.

We report the cloning of the gyrB gene from Streptococcus pneumoniae 533 that carries the nov-1 allele. The gyrB gene codes for a protein homologous to the gyrase B subunit of archaebacteria and eubacteria. The same amino acid substitution (Ser-127 to Leu) confers novobiocin resistance on four isolates of S. pneumoniae. This amino acid position is equivalent to Val-120 of Escherichia coli GyrB, a residue that lies inside the ATP-binding domain as revealed by the crystal structure of the protein.

ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of fluoroquinolones and cooperates with DNA gyrase A subunit in forming resistance phenotype.

The genes encoding the ParC and ParE subunits of topoisomerase IV of Streptococcus pneumoniae, together with the region encoding amino acids 46 to 172 (residue numbers are as in Escherichia coli) of the pneumococcal GyrA subunit, were partially characterized. The gyrA gene maps to a physical location distant from the gyrB and parC loci on the chromosome, whereas parC is closely linked to parE. Ciprofloxacin-resistant (Cpr) clinical isolates of S. pneumoniae had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level Cpr) or in both quinolone resistance-determining regions of ParC and GyrA (high-level Cpr). Mutations were found in residue positions equivalent to the serine at position 83 and the aspartic acid at position 87 of the E. coli GyrA subunit. Transformation experiments suggest that ParC is the primary target of ciprofloxacin. Mutation in parC appears to be a prerequisite before mutations in gyrA can influence resistance levels.

Quinine specifically inhibits the proteolipid subunit of the F0F1 H+ -ATPase of Streptococcus pneumoniae.

Streptococcus pneumoniae is uniquely sensitive to quinine and its derivatives, but only those alkaloids having antimalarial properties, i.e., those in the erythro configuration, also possess antipneumococcal activity. Quinine and related compounds inhibit the pneumococcal H+ -ATPase. Quinine- and optochin-resistant pneumococci showed mutations that change amino acid residues located in one of the two transmembrane alpha-helices of the c subunit of the F0F1, H+ -ATPase.

Molecular basis of the optochin-sensitive phenotype of pneumococcus: characterization of the genes encoding the F0 complex of the Streptococcus pneumoniae and Streptococcus oralis H(+)-ATPases.

The gene responsible for the optochin-sensitive (OptS) phenotype of Streptococcus pneumoniae has been characterized. Sequence comparisons indicated that the genes involved encoded the subunits of the F0 complex of an H(+)-ATPase. Sequence analysis and transformation experiments showed that the atpC gene is responsible for the optochin-sensitive resistant (OptS/OptR) phenotype. Our results also show that natural as well as laboratory OptR isolates have arisen by point mutations that produce different amino acid changes at positions 48, 49 or 50 of the ATPase c subunit. The nucleotide sequence of the F0 complex of the Streptococcus oralis ATPase has also been determined. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (OptS) and M222 (an OptR strain produced by interspecies recombination between pneumococcus and S. oralis), and S. oralis revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin specifically inhibited the membrane-bound ATPase activity of the S. pneumoniae wild-type (OptS) strains, and found a 100-fold difference between OptS and OptR strains, both in growth inhibition and in membrane ATPase resistance.

Proteins encoded by the DpnI restriction gene cassette. Hyperproduction and characterization of the DpnI endonuclease.

Insertion mutations in the DpnI gene cassette of Streptococcus pneumoniae indicated that the two genes it contains, dpnC and dpnD, were transcribed from an adjacent promoter and that only dpnC was necessary for expression of the DpnI endonuclease. Large amounts of the DpnI endonuclease were produced from the cloned cassette in an Escherichia coli expression system, and the enzyme was purified to homogeneity. The DpnI endonuclease is composed of a single polypeptide of 30 kDa, which, as shown by NH2-terminal sequencing of the protein, is encoded by the entire dpnC open reading frame. The native protein sedimented as a monomer of 30 kDa in 0.5 M NaCl. A protein composed of a 20-kDa polypeptide, which is presumably encoded by dpnD, was also produced in large amounts. It was partially purified, but its function is unknown. Examination of the predicted amino acid sequence of DpnI revealed a potential metal-containing, DNA-binding finger structure. It is suggested that this structure provides the specificity for recognition of the methylated DNA sequence, 5'-GmATC-3', that is cleaved by the DpnI endonuclease.

Three regions in the DNA of plasmid pLS1 show sequence-directed static bending.

Three regions showing abnormal electrophoretic mobility, which is an indication of the existence of bends in DNA, have been observed in the DNA of plasmid pLS1. These loci have been characterized by assays designed to detect sequence-directed bending in DNA (temperature-dependence migration and two dimensional electrophoresis). The first region (locus B-1) was located within a fragment that contains a proposed inhibitor countertranscribed RNA (RNAII). The second locus (B-2) contains the plasmid plus origin of replication and the third region (locus B-3) was located in the vicinity of a putative antisense RNA (RNAI) of unknown function. The centres of the first two bent DNA regions were located by circular permutation assays at nucleotides 882 (locus B-1) and 634 (locus B-2). The bend centre of locus B-1 was found to be upstream of the promoter for the putative antisense RNAII. The centre of curvature in locus B-2 was located in the vicinity of the putative promoter of the replication proteins RepA and RepB and of a sequence that has three 11-bp direct repeats. The DNA sequence at this region showed the existence of A.T tracts, with an internal repeat of 10 to 11 base pairs, for five helix turns. A complex curvature in the DNA of pLS1 at locus B-2 that may have a regulatory role in plasmid replication is postulated.

Regulation of competence for genetic transformation in Streptococcus pneumoniae: expression of dpnA, a late competence gene encoding a DNA methyltransferase of the DpnII restriction system.

The chromosomal DpnII gene cassette of Streptococcus pneumoniae encodes two methyltransferases and an endonuclease. One methyltransferase acts on double-stranded and the other on single-stranded DNA. Two mRNAs are transcribed from the cassette. One, a SigA promoter transcript, includes all three genes; the other includes a truncated form of the second methyltransferase gene (dpnA) and the endonuclease gene. The truncated dpnA, which is translated from the second start codon in the full gene, was shown to produce active enzyme. A promoter reporter plasmid for S. pneumoniae was devised to characterize the promoter for the second mRNA. This transcript was found to depend on a promoter that responded to the induction of competence for genetic transformation. The promoter contains the combox sequence recognized by a SigH-containing RNA polymerase. As part of the competence regulon, the dpnA gene makes a product able to methylate incoming plasmid strands to protect them from the endonuclease and allow plasmid establishment. Its function differs from most genes in the regulon, which are involved in DNA uptake. Comparison of R6 and Rx strains of S. pneumoniae showed the temperature dependence of transformation in R6 to result from temperature sensitivity of the uptake apparatus and not the development of competence.

Horizontal transfer of parC and gyrA in fluoroquinolone-resistant clinical isolates of Streptococcus pneumoniae.

We have analyzed genetically three clinical isolates (3180, 3870, and 1244) of Streptococcus pneumoniae with high-level ciprofloxacin resistance. Isolates 3180 and 3870 were atypical because of their insolubility in deoxycholate. However, they hybridized specifically with pneumococcal autolysin and pneumolysin gene probes and have typical pneumococcal atpC and atpA gene sequences. Analysis of the complete sequences of the parC and gyrA genes revealed total variations of 8 and 8.7% (isolate 3180) and 7.4 and 3.6% (isolate 3870), respectively, compared to the wild-type strain R6 sequence. The variations observed between the sequences of R6 and isolate 1244 were less than 0.9%. The structure of the gyrA and parC genes from isolates 3180 and 3870 was organized in sequence blocks that show different levels of divergence, suggesting a pattern of recombination. These results are evidence for recombination at the fluoroquinolone target genes in clinical isolates of S. pneumoniae. The genetically related viridans group streptococci could act as a reservoir for fluoroquinolone resistance genes.

[Streptococcus salivarius meningitis after spinal anesthesia]. / Meningitis por Streptococcus salivarius tras anestesia espinal.

Streptococcus salivarius is a usual commensal of skin, gastrointestinal tract, genitourinary tract, oral cavity and paranasal sinuses. Although it is usually considered to have low virulence, S. salivarius may cause life-threatening infections, particularly endocarditis. On the other hand, bacterial meningitis after spinal anesthesia is very rare, there being some reported cases caused by S. salivarius, S. mitis, Staphylococcus aureus and Enterococcus faecalis. We report a 57 year old man who developed meningitis symptoms within 10 h of an uncomplicated inguinal herniorrhaphy performed during spinal anesthesia. Cerebrospinal cultures grew S. salivarius sensitive to penicillin. The patient was successfully treated with penicillin G and left the hospital without sequelae. In the literature, bacterial meningitis due to S. salivarius is rarely reported. Of the 28 cases, 18 occurred after lumbar puncture for diagnostic or for spinal anesthesia, 5 occurred following a bacteriemia for upper gastrointestinal endoscopy or intestinal neoplasia, and the other 5 in patients who had dural defects. We discuss the possible etiological causes of the meningitis due to S. salivarius cases reports. The early recognition of this entity and the aseptic precautions likely to reduce the incidence of infectious complications after lumbar puncture are stressed.

Coupling between DNA replication and cell division mediated by the FtsA protein in Escherichia coli: a pathway independent of the SOS response, the "TER" pathway.

Inhibition of DNA synthesis prevented the recovery of cell division in filaments of D-3R [ftsA3(Ts) recA56] returned to the permissive temperature. The FtsA protein may be a signal involved in the "TER" pathway, a series of events that coordinate cell division with DNA replication, that is independent of the SOS pathway.

Co-ordination between elongation and division in Escherichia coli mediated by the wee gene product.

A procedure to construct strains of Escherichia coli containing conditional lethal mutations in two different genes was used to construct a ftsA-3(ts) wee(Am) supF(ts) strain. This strain, OV-25-7, was used to ascertain whether the wee gene product (Wee) acts at the level of regulation of cell elongation or at the co-ordination of elongation and division. The mass per unit length and the buoyant density of cells in the absence of Wee increased only if division was allowed, as in the case of strain OV-25 (wee(Am) supF(ts)), but not when it was inhibited, as in strain OV-25-7. These results suggested that in E. coli the wee gene product was acting at the level of coordination between elongation and cell division.

Molecular characterization of the gene encoding the DNA gyrase A subunit of Streptococcus pneumoniae.

The gene encoding the DNA gyrase A subunit of Streptococcus pneumoniae was cloned and sequenced. The gyrA gene codes for a protein of 822 amino acids homologous to the gyrase A subunit of eubacteria. Translation of the gene in an Escherichia coli expression system revealed a 92-kDa polypeptide. A sequence-directed DNA curvature was identified in the promoter region of gyrA. The bend center was mapped and located between the -35 and -10 regions of the promoter. Primer extension analysis showed that gyrA transcription initiates 6 bp downstream of an extended -10 promoter. The possible implications of the bent DNA region as a regulatory element in the transcription of gyrA are discussed.

The promoter of the operon encoding the F0F1 ATPase of Streptococcus pneumoniae is inducible by pH.

The genes encoding the subunits of the F0F1 membrane ATPase of Streptococcus pneumoniae were cloned and sequenced. The eight genes, transcribed to one mRNA, are organized in an operon encoding the c, a, b, delta, alpha, gamma, beta and epsilon subunits of 66, 238, 165, 178, 501, 292, 471 and 139 amino acid residues, respectively, that were expressed in an Escherichia coli system. To investigate the role of the ATPase in the regulation of the intracellular pH, the expression of the operon between pH 5.7 and 7.5 was studied. An increase in both the ATPase activity and the amount of the alpha and beta F1 subunits as shown by Western blot analysis was observed as the pH decreased. These increases were accompanied by an increase in the atp-specific mRNA, as shown by Northern blot and slot-blot analysis. Primer extension experiments and transcriptional fusions between the atp promoter and the reporter cat gene demonstrated that this pH-dependent increase in the mRNA was regulated at the level of initiation of transcription. Transcription of the operon occurs from a promoter with a consensus -35 box (TTGACA) and a -10 box (TACACT) that differs from the consensus (TATAAT). A point mutation at the -10 box of the promoter (change to TGCACT) avoided this increase, suggesting a role for this sequence in the pH-inducible regulation.