Biological Effects of Quinolones: A Family of Broad-Spectrum Antimicrobial Agents.

Broad antibacterial spectrum, high oral bioavailability and excellent tissue penetration combined with safety and few, yet rare, unwanted effects, have made the quinolones class of antimicrobials one of the most used in inpatients and outpatients. Initially discovered during the search for improved chloroquine-derivative molecules with increased anti-malarial activity, today the quinolones, intended as antimicrobials, comprehend four generations that progressively have been extending antimicrobial spectrum and clinical use. The quinolone class of antimicrobials exerts its antimicrobial actions through inhibiting DNA gyrase and Topoisomerase IV that in turn inhibits synthesis of DNA and RNA. Good distribution through different tissues and organs to treat Gram-positive and Gram-negative bacteria have made quinolones a good choice to treat disease in both humans and animals. The extensive use of quinolones, in both human health and in the veterinary field, has induced a rise of resistance and menace with leaving the quinolones family ineffective to treat infections. This review revises the evolution of quinolones structures, biological activity, and the clinical importance of this evolving family. Next, updated information regarding the mechanism of antimicrobial activity is revised. The veterinary use of quinolones in animal productions is also considered for its environmental role in spreading resistance. Finally, considerations for the use of quinolones in human and veterinary medicine are discussed.

Structural changes and differentially expressed genes in Pseudomonas aeruginosa exposed to meropenem-ciprofloxacin combination.

The effect of a meropenem-ciprofloxacin combination (MCC) on the susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa (MRPA) clinical isolates was determined using checkerboard and time-kill curve techniques. Structural changes and differential gene expression that resulted from the synergistic action of the MCC against one of the P. aeruginosa isolates (1071-MRPA]) were evaluated using electron microscopy and representational difference analysis (RDA), respectively. The differentially expressed, SOS response-associated, and resistance-associated genes in 1071-MRPA exposed to meropenem, ciprofloxacin, and the MCC were monitored by quantitative PCR. The MCC was synergistic against 25% and 40.6% of MDR P. aeruginosa isolates as shown by the checkerboard and time-kill curves, respectively. The morphological and structural changes that resulted from the synergistic action of the MCC against 1071-MRPA were a summation of the effects observed with each antimicrobial alone. One exception included outer membrane vesicles, which were seen in a greater amount upon ciprofloxacin exposure but were significantly inhibited upon MCC exposure. Cell wall- and DNA repair-associated genes were differentially expressed in 1071-MRPA exposed to meropenem, ciprofloxacin, and the MCC. However, some of the RDA-detected, resistance-associated, and SOS response-associated genes were expressed at significantly lower levels in 1071-MRPA exposed to the MCC. The MCC may be an alternative for the treatment of MDR P. aeruginosa. The effect of this antimicrobial combination may be not only the result of a summation of the effects of meropenem and ciprofloxacin but also a result of differential action that likely inhibits protective mechanisms in the bacteria.

Influence of pH and temperature on the expression of sboA and ituD genes in Bacillus sp. P11.

Temperature and pH are key factors influencing the production of antimicrobial peptides. In this work, qRT-PCR methodology was used to demonstrate the effect of these two variables on sboA (subtilosin A) and ituD (iturin A) expression in Bacillus sp. P11, an isolate from aquatic environment of the Amazon. Bacillus sp. P11 was incubated in BHI broth for 36 h at 30, 37 and 42 °C, and the pH values were 6.0, 7.4 and 8.0. The production of subtilosin A and iturin A was confirmed by mass spectrometry. The sboA expression increased 200-fold when the initial pH was 8.0. In contrast, ituD expression was maximum at pH 6.0. Increased temperature (42 °C) was adverse for both genes, but ituD expression increased at 37 °C. Expression of sboA and ituD was strongly affected by pH and temperature and qRT-PCR proved to be a powerful tool to investigate the potential of Bacillus strains to produce subtilosin A and iturin A.

Pseudomonas aeruginosa PA14 cupD transcription is activated by the RcsB response regulator, but repressed by its putative cognate sensor RcsC.

The opportunistic pathogen Pseudomonas aeruginosa PA14 possesses four fimbrial cup clusters, which may confer the ability to adapt to different environments. cupD lies in the pathogenicity island PAPI-1 next to genes coding for a putative phosphorelay system composed of the hybrid histidine kinase RcsC and the response regulator RcsB. The main focus of this work was the regulation of cupD at the mRNA level. It was found that the HN-S-like protein MvaT does not exert a strong influence on cupD transcript levels, as it does for cupA. cupD transcription is higher in cultures grown at 28 degrees C, which agrees with a cupD mutant presenting attenuated virulence only in a plant model, but not in a mouse model of infection. Whereas an rcsC in-frame deletion mutant presented higher levels of cupD mRNA, rcsB deletion had the opposite effect. Accordingly, overexpression of RcsB increased the levels of cupD transcription, and promoted biofilm formation and the appearance of fimbriae. A single transcription start site was determined for cupD and transcription from this site was induced by RcsB. A motif similar to the enterobacterial RcsB/RcsA-binding site was detected adjacent to the -35 region, suggesting that this could be the RcsB-binding site. Comparison of P. aeruginosa and Escherichia coli Rcs may provide insights into how similar systems can be used by different bacteria to control gene expression and to adapt to various environmental conditions.

Pet secretion, internalization and induction of cell death during infection of epithelial cells by enteroaggregative Escherichia coli.

In an in vitro model using HEp-2 cells treated with purified plasmid-encoded toxin (Pet), we have identified morphological changes characterized by cell rounding and detachment after toxin internalization; these changes progress to cell death. However, these effects have not yet been shown to occur during the infection of epithelial cells by enteroaggregative Escherichia coli (EAEC). Here, we show that the secretion of Pet by EAEC is regulated at the transcriptional level, since secretion was inhibited in eukaryotic cell culture medium, although Pet was efficiently secreted in the same medium supplemented with tryptone. Inefficient secretion of Pet by EAEC in DMEM prevented cell detachment, whereas efficient Pet secretion in DMEM/tryptone increased cell detachment in a HEp-2 cell adherence assay. Interestingly, Pet toxin was efficiently delivered to epithelial cells, since it was internalized into epithelial cells infected with EAEC at similar concentrations to those obtained by using 37 microg ml(-1) purified Pet protein. Additionally, Pet was not internalized when the epithelial cells were infected with a pet clone, HB101(pCEFN1), unlike the wild-type strain, which has a high adherence capability. There is a correlation between Pet secretion by EAEC, the internalization of Pet into epithelial cells, cell detachment and cell death in EAEC-infected cells. The ratio between live and dead cells decreased in cells treated with wild-type EAEC in comparison with cells treated with an isogenic mutant in the pet gene, whereas the effects were restored by complementing the mutant with the pet gene. All these data indicate that Pet is an important virulence factor in the pathogenesis of EAEC infection.

Acidic stress induces autolysis by a CSP-independent ComE pathway in Streptococcus pneumoniae.

In Streptococcus pneumoniae, autolysis is considered a programmed cell-death process executed principally by the major autolysin (LytA), and the underlying mechanism causing its activation is not completely understood. It is known that autolysis is triggered by competence development at alkaline pH and regulated by a two-component system, ComDE, which senses a competence-stimulating peptide (CSP) and behaves as a quorum-sensing mechanism. In this work, we found that acidic stress triggered a LytA-mediated autolysis and, curiously, this phenomenon was regulated by a CSP-independent ComE pathway. A further analysis of a hyperactive ComD mutant revealed that ComE needs to be phosphorylated to activate acidic stress-induced lysis (ASIL). The comE transcripts were induced by acidic culture conditions, suggesting that ComE could be sensing acidic stress. We also investigated CiaRH, a two-component system whose null mutants show a comE derepression and a CSP-dependent autolysis induction at alkaline pH. By analysis of cia comE double mutants, we demonstrated that CiaRH protected cells from ASIL by a ComE-independent pathway. Here, we propose that ComE is the principal route of the signalling pathway that determines a global stress response, and clearly regulates the induction of the LytA-mediated programmed cell death in S. pneumoniae. Acidic stress may represent for S. pneumoniae an alternative condition, in addition to competence and antibiotics, to assure the release of virulence factors, DNA and cell-wall compounds by autolysis, favouring genetic exchange and contributing to its pathogenesis.

Study of the role of Mce3R on the transcription of mce genes of Mycobacterium tuberculosis.

BACKGROUND: mce3 is one of the four virulence-related mce operons of Mycobacterium tuberculosis. In a previous work we showed that the overexpression of Mce3R in Mycobacterium smegmatis and M. tuberculosis abolishes the expression of lacZ fused to the mce3 promoter, indicating that Mce3R represses mce3 transcription. RESULTS: We obtained a knockout mutant strain of M. tuberculosis H37Rv by inserting a hygromycin cassette into the mce3R gene. The mutation results in a significant increase in the expression of mce3 genes either in vitro or in a murine cell macrophages line as it was determined using promoter-lacZ fusions in M. tuberculosis. The abundance of mce1, mce2 and mce4 mRNAs was not affected by this mutation as it was demonstrated by quantitative RT-PCR. The mce3R promoter activity in the presence of Mce3R was significantly reduced compared with that in the absence of the regulator, during the in vitro culture of M. tuberculosis. CONCLUSION: Mce3R repress the transcription of mce3 operon and self regulates its own expression but does not affect the transcription of mce1, mce2 and mce4 operons of M. tuberculosis.

Characterization of a novel and genetically different small infective variant of Piscirickettsia salmonis.

We report a novel genetically different small infective variant of the fish pathogen Piscirickettsia salmonis (sP.s). sP.s variants were recovered both from ageing post-infected CHSE-214 culture cells as well as from naturally infected fish. The ITS region of sP.s variants, although sharing a common core sequence, is different from the ITS of the prototype strain LF-89 from which they originate. Thus, sP.s can be selectively amplified with sequence-specific discriminatory set of PCR primers. Transcriptionally, sP.s are fully active as shown by reverse transcription PCR analysis. Immunologically, sP.s is specifically recognized by antibodies against standard P. salmonis. Structurally, atomic force microscopy shows that sP.s. is well below (<0.2microm) the standard range size described for this pathogen (0.5-1.5microm). Functionally, although sP.s is infective their in vitro progeny is a hundred percent identical to the LF-89 prototype strain. In summary sP.s, represent selectable infective variants of the LF-89 strain and not new strains, probably resulting from a survival strategy of the bacteria in response to limiting growth conditions. In this frame, sP.s could be responsible of horizontal infection of fish in the field.

O-antigen modal chain length in Shigella flexneri 2a is growth-regulated through RfaH-mediated transcriptional control of the wzy gene.

Shigella flexneri 2a 2457T produces lipopolysaccharide (LPS) with two O-antigen (OAg) chain lengths: a short (S-OAg) controlled by WzzB and a very long (VL-OAg) determined by Wzz(pHS-2). This study demonstrates that the synthesis and length distribution of the S. flexneri OAg are under growth-phase-dependent regulation. Quantitative electrophoretic analysis showed that the VL-OAg increased during growth while the S-OAg distribution remained constant. Increased production of VL-OAg correlated with the growth-phase-regulated expression of the transcription elongation factor RfaH, and was severely impaired in a DeltarfaH mutant, which synthesized only low-molecular-mass OAg molecules and a small amount of S-OAg. Real-time RT-PCR revealed a drastic reduction of wzy polymerase gene expression in the DeltarfaH mutant. Complementation of this mutant with the wzy gene cloned into a high-copy-number plasmid restored the bimodal OAg distribution, suggesting that cellular levels of Wzy influence not only OAg polymerization but also chain-length distribution. Accordingly, overexpression of wzy in the wild-type strain resulted in production of a large amount of high-molecular-mass OAg molecules. An increased dosage of either wzzB or wzz(pHS-2) also altered OAg chain-length distribution. Transcription of wzzB and wzz(pHS-2) genes was regulated during bacterial growth but in an RfaH-independent manner. Overall, these findings indicate that expression of the wzy, wzzB and wzz(pHS-2) genes is finely regulated to determine an appropriate balance between the proteins responsible for polymerization and chain-length distribution of S. flexneri OAg.

Bacterial toxicity of potassium tellurite: unveiling an ancient enigma.

Biochemical, genetic, enzymatic and molecular approaches were used to demonstrate, for the first time, that tellurite (TeO(3) (2-)) toxicity in E. coli involves superoxide formation. This radical is derived, at least in part, from enzymatic TeO(3) (2-) reduction. This conclusion is supported by the following observations made in K(2)TeO(3)-treated E. coli BW25113: i) induction of the ibpA gene encoding for the small heat shock protein IbpA, which has been associated with resistance to superoxide, ii) increase of cytoplasmic reactive oxygen species (ROS) as determined with ROS-specific probe 2'7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA), iii) increase of carbonyl content in cellular proteins, iv) increase in the generation of thiobarbituric acid-reactive substances (TBARs), v) inactivation of oxidative stress-sensitive [Fe-S] enzymes such as aconitase, vi) increase of superoxide dismutase (SOD) activity, vii) increase of sodA, sodB and soxS mRNA transcription, and viii) generation of superoxide radical during in vitro enzymatic reduction of potassium tellurite.

Microcin J25 uptake: His5 of the MccJ25 lariat ring is involved in interaction with the inner membrane MccJ25 transporter protein SbmA.

Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide consisting of 21 L-amino acid residues (G1-G-A-G-H5-V-P-E-Y-F10-V-G-I-G-T15-P-I-S-F-Y20-G). E. coli RNA polymerase (RNAP) is the intracellular target of MccJ25. MccJ25 enters cells after binding to specific membrane transporters: FhuA in the outer membrane and SbmA in the inner membrane. Here, we studied MccJ25 mutants carrying a substitution of His5 by Lys, Arg, or Ala. The inhibitory effects on cellular growth and in vitro RNAP activity were determined for each mutant microcin. The results show that all mutants inhibited RNAP in vitro. However, the mutants were defective in their ability to inhibit cellular growth. Experiments in which the FhuA protein was bypassed showed that substitutions of MccJ25 His5 affected the SbmA-dependent transport. Our results thus suggest that MccJ25 His5 located in the lariat ring is involved, directly or indirectly, in specific interaction with SbmA and is not required for MccJ25 inhibition of RNAP.

Molecular basis of thermosensing: a two-component signal transduction thermometer in Bacillus subtilis.

Both prokaryotes and eukaryotes respond to a decrease in temperature with the expression of a specific subset of proteins. Although a large body of information concerning cold shock-induced genes has been gathered, studies on temperature regulation have not clearly identified the key regulatory factor(s) responsible for thermosensing and signal transduction at low temperatures. Here we identified a two-component signal transduction system composed of a sensor kinase, DesK, and a response regulator, DesR, responsible for cold induction of the des gene coding for the Delta5-lipid desaturase from Bacillus subtilis. We found that DesR binds to a DNA sequence extending from position -28 to -77 relative to the start site of the temperature-regulated des gene. We show further that unsaturated fatty acids (UFAs), the products of the Delta5-desaturase, act as negative signalling molecules of des transcription. Thus, a regulatory loop composed of the DesK-DesR two-component signal transduction system and UFAs provides a novel mechanism for the control of gene expression at low temperatures.

Bacteriostatic action of streptomycin on ribosomally resistant mutants (rpsL) of Salmonella typhimurium.

Incubation of streptomycin-resistant (rpsL) mutants of Salmonella typhimurium in alkaline nutrient medium containing streptomycin brought about an inhibition of cell growth that was readily reversed by removing the antibiotic or neutralizing the medium. Growth inhibition was maximal at pH 8.2 and a streptomycin concentration of 800 micrograms/ml. A similar amount of dihydrostreptomycin had a negligible effect, and 10-times-higher concentrations of this antibiotic were required to reproduce the streptomycin action. Addition of streptomycin (400 micrograms/ml) to rpsL cells in alkaline (pH 8.2) nutrient medium caused inhibition of protein and DNA synthesis and also, but to a lower degree, of RNA synthesis. This effect on macromolecular synthesis was not due to ATP deprivation, since ATP content rose after addition of the antibiotic. At pH 8.2, the rate of entrance of streptomycin increased fourfold with respect to the rate at pH 7.0, leading to a large accumulation of streptomycin into rpsL cells. Uptake of the antibiotic was halted by addition of KCN or chloramphenicol. Equal uptake was obtained with 800 micrograms of dihydrostreptomycin or 400 micrograms of streptomycin per ml, yet the former did not affect cell growth at that concentration. It is concluded that high pH stimulates streptomycin and dihydrostreptomycin uptake by rpsL strains but only streptomycin accumulation causes growth inhibition in cells lacking the high-affinity ribosomal site.

What is an antibiotic?

Potential sites of action are innumerable for selective toxicity of antibiotics to bacterial cells and not to host cells. Available antibiotics are effective by inhibiting synthesis of cell walls, protein, RNA, DNA, or folic acid. Both selective toxicity of antibiotics and resistance to antibiotics depend on the site of action and delivery to the site of action in adequate concentrations. Critical mechanisms for the transport of antibiotics through bacterial barriers and important pharmacokinetic attributes in humans are involved. Adverse effects of antibiotics may be immunologic or nonimmunologic. Nonimmunologic adverse effects of antibiotics are rarely related to slippage in the selectivity of toxicity but are usually the result of unrelated biochemical effects associated with other undesired actions of all drugs. Such undesired side effects can be avoided in drug development only if we understand the biochemical basis of these actions.

Concepts of function of peripheral non-chromatin and endosome in Entamoeba histolytica.

The distribution of RNA (to a lesser extent DNA) in the nucleus of E. histolytica appeared to be the opposite of what one found in other eukaryotic cells (originally stated by Pan and Geiman in 1955). Autoradiographs of amebae labelled with 3H-thymidine revealed primarily randomly distributed DNA-3H (probably euchromatin) in interphase cells. This DNA was so low in amount in individual cells that it usually did not stain with the Feulgen procedure. A small amount of DNA-3H was also detected by autoradiography in the peripheral chromatin (= rDNA?; occasionally it was abundant) and endosome. Clumps of DNA-3H were also observed around the endosomal area in some amebae. This labelling pattern was not observed in 3H-uridine (for RNA) incorporation experiments. These data were consistent with the possibility that the endosome, which was sometimes Feulgen +, was a site of condensation of DNA prior to nuclear division. As detected by light and electron microscopy autoradiographic analysis of 3H-uridine incorporation, RNA was synthesized (or accumulated) primarily in the peripheral chromatin; lesser amounts were evenly distributed. Most of the peripherally located amebal nuclear RNA was probably rRNA and/or precursor rRNA. If this is the case, it is probable that the peripheral chromatin, although structurally unlike nucleoli, may be "functionally" comparable to nucleoli of eukaryotes.