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Spreading resistance to antibiotics and the emergence of multidrug-resistant strains have become frequent in many bacterial species, including mycobacteria, which are the causative agents of severe diseases and which have profound impacts on global health. Here, we used a system of microfluidics, fluorescence microscopy, and target-tagged fluorescent reporter strains of Mycobacterium smegmatis to perform real-time monitoring of replisome and chromosome dynamics following the addition of replication-altering drugs (novobiocin, nalidixic acid, and griselimycin) at the single-cell level. We found that novobiocin stalled replication forks and caused relaxation of the nucleoid and that nalidixic acid triggered rapid replisome collapse and compaction of the nucleoid, while griselimycin caused replisome instability, with the subsequent overinitiation of chromosome replication and overrelaxation of the nucleoid. In addition to study target-drug interactions, our system also enabled us to observe how the tested antibiotics affected the physiology of mycobacterial cells (i.e., growth, chromosome segregation, etc.)., (Copyright © 2019 Trojanowski et al.)

DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1× MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin concentrations greater than 1× MIC. When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (∼150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1× and 1.7× MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrA mutation on accumulation of gyrase covalent complexes on chromosomal DNA.

Antibiotic TA is a macrocyclic secondary metabolite produced by myxobacteria that has broad-spectrum bactericidal activity. The structure of TA is unique, and its molecular target is unknown. Here, we sought to elucidate TA's mode of action (MOA) through two parallel genetic approaches. First, chromosomal Escherichia coli TA-resistant mutants were isolated. One mutant that showed specific resistance toward TA was mapped and resulted from an IS4 insertion in the lpp gene, which encodes an abundant outer membrane (Braun's) lipoprotein. In a second approach, the comprehensive E. coli ASKA plasmid library was screened for overexpressing clones that conferred TA(r). This effort resulted in the isolation of the lspA gene, which encodes the type II signal peptidase that cleaves signal sequences from prolipoproteins. In whole cells, TA was shown to inhibit Lpp prolipoprotein processing, similar to the known LspA inhibitor globomycin. Based on genetic evidence and prior globomycin studies, a block in Lpp expression or prevention of Lpp covalent cell wall attachment confers TA(r) by alleviating a toxic buildup of mislocalized pro-Lpp. Taken together, these data argue that LspA is the molecular target of TA. Strikingly, the giant ta biosynthetic gene cluster encodes two lspA paralogs that we hypothesize play a role in producer strain resistance.

Subinhibitory concentrations of some antibiotics, such as fluoroquinolones, have been reported to stimulate mutation and, consequently, bacterial adaptation to different stresses, including antibiotic pressure. In Escherichia coli, this stimulation is mediated by alternative DNA polymerases induced via the SOS response. Sublethal concentrations of the fluoroquinolone ciprofloxacin have been shown to stimulate recombination between divergent sequences in E. coli. However, the effect of ciprofloxacin on recombination between homologous sequences and its SOS dependence have not been studied. Moreover, the possible effects of other antibiotics on homologous recombination remain untested. The aim of this work was to study the effects of sublethal concentrations of ciprofloxacin and 10 additional antibiotics, including different molecular families with different molecular targets, on the rate of homologous recombination of DNA in E. coli. The antibiotics tested were ciprofloxacin, ampicillin, ceftazidime, imipenem, chloramphenicol, tetracycline, gentamicin, rifampin (rifampicin), trimethoprim, fosfomycin, and colistin. Our results indicate that only ciprofloxacin consistently stimulates the intrachromosomal recombinogenic capability of homologous sequences in E. coli. The ciprofloxacin-based stimulation occurs at concentrations and times that apparently do not dramatically compromise the viability of the whole population, and it is dependent on RecA and partially dependent on SOS induction. One of the main findings of this work is that, apart from quinolone antibiotics, none of the most used antibiotics, including trimethoprim (a known inducer of the SOS response), has a clear side effect on homologous recombination in E. coli. In addition to the already described effects of some antibiotics on mutagenicity, DNA transfer, and genetic transformability in naturally competent species, the effect of increasing intrachromosomal recombination of homologous DNA sequences can be uniquely ascribed to fluoroquinolones, at least for E. coli.

Klebsiella pneumoniae causes common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance. The emergence and spread of beta-lactamase-producing K. pneumoniae strains highlight the need to develop new therapeutic strategies. In this study, we developed antisense peptide nucleic acids (PNAs) conjugated to the (KFF)(3)K peptide and investigated whether they could mediate gene-specific antisense effects in K. pneumoniae. No outer membrane permeabilization was observed with antisense PNAs when used alone. Antisense peptide-PNAs targeted at two essential genes, gyrA and ompA, were found to be growth inhibitory at concentrations of 20 muM and 40 muM, respectively. Mismatched antisense peptide-PNAs with sequence variations of the gyrA and ompA genes when used as controls were not growth inhibitory. Bactericidal effects exerted by peptide-anti-gyrA PNA and peptide-anti-ompA PNA on cells were observed within 6 h of treatment. The antisense peptide-PNAs specifically inhibited expression of DNA gyrase subunit A and OmpA from the respective targeted genes in a dose-dependent manner. Both antisense peptide-PNAs cured IMR90 cell cultures that were infected with K. pneumoniae (10(4) CFU) in a dose-dependent manner without any noticeable toxicity to the human cells.

Aminoglycosides bind to the 16S rRNA at the tRNA acceptor site (A site) and disturb protein synthesis by inducing codon misreading. We investigated Escherichia coli cell elongation and division, as well as the dynamics of chromosome replication and segregation, in the presence of sublethal concentrations of amikacin (AMK). The fates of the chromosome ori and ter loci were monitored by visualization by using derivatives of LacI and TetR fused to fluorescent proteins in E. coli strains that carry operator arrays at the appropriate locations. The results showed that cultures containing sublethal concentrations of AMK contained abnormally elongated cells. The chromosomes in these cells were properly located, suggesting that the dynamics of replication and segregation were normal. FtsZ, an essential protein in the process of cell division, was studied by using an ectopic FtsZ-cyan fluorescent protein fusion. Consistent with a defect in cell division, we revealed that the Z ring failed to properly assemble in these elongated cells.

A new gene, sul3, which specifies a 263-amino-acid protein similar to a dihydropteroate synthase encoded by the 54-kb conjugative plasmid pVP440 from Escherichia coli was characterized. Expression of the cloned sul3 gene conferred resistance to sulfamethoxazole on E. coli. Two copies of the insertion element IS15Delta/26 flanked the region containing sul3. The sul3 gene was detected in one-third of the sulfonamide-resistant pathogenic E. coli isolates from pigs in Switzerland.

A clinical isolate of Pseudomonas aeruginosa was found to produce a clavulanic acid-inhibited extended-spectrum beta-lactamase with a pI of 6.4. PCR, cloning, and sequencing experiments showed that the corresponding bla(TEM-21) gene was part of a chromosomally located Tn801 transposon disrupted by an IS6100 element and adjacent to an aac(3)-II gene.

The effect of quinolones on the inhibition of DNA synthesis in Staphylococcus aureus was examined by using single resistance mutations in parC or gyrA to distinguish action against gyrase or topoisomerase IV, respectively. Norfloxacin preferentially attacked topoisomerase IV and blocked DNA synthesis slowly, while nalidixic acid targeted gyrase and inhibited replication rapidly. Ciprofloxacin exhibited an intermediate response, consistent with both enzymes being targeted. The absence of RecA had little influence on target choice by this assay, indicating that differences in rebound (repair) DNA synthesis were not responsible for the results. At saturating drug concentrations, norfloxacin and a gyrA mutant were used to show that topoisomerase IV-norfloxacin-cleaved DNA complexes are distributed on the S. aureus chromosome at intervals of about 30 kbp. If cleaved complexes block DNA replication, as indicated by previous work, such close spacing of topoisomerase-quinolone-DNA complexes should block replication rapidly (replication forks are likely to encounter a cleaved complex within a minute). Thus, the slow inhibition of DNA synthesis at growth-inhibitory concentrations suggests that a subset of more distantly distributed complexes is physiologically relevant for drug action and is unlikely to be located immediately in front of the DNA replication fork.

The aarE1 allele was identified on the basis of the resulting phenotype of increased aminoglycoside resistance. The aarE1 mutation also resulted in a small-colony phenotype and decreased levels of aac(2')-Ia mRNA. The deduced AarE gene product displayed 61% amino acid identity to the Escherichia coli UbiA protein, an octaprenyltransferase required for the second step of ubiquinone biosynthesis. Complementation experiments in both Providencia stuartii and E. coli demonstrated that aarE and ubiA are functionally equivalent.

Many strains of Pseudomonas aeruginosa are resistant to the antibiotics cerulenin and thiolactomycin, potent inhibitors of bacterial fatty acid biosynthesis. A novel yeast Flp recombinase-based technique was used to isolate an unmarked mexAB-oprM deletion encoding an efflux system mediating resistance to multiple antibiotics in P. aeruginosa. The experiments showed that the MexAB-OprM system is responsible for the intrinsic resistance of this bacterium to cerulenin and thiolactomycin. Whereas thiolactomycin was not a substrate of the MexCD-OprJ pump expressed in a delta(mexAB-oprM) nfxB mutant, cerulenin was efficiently effluxed by the MexCD-OprJ system. It was also found that the MexAB-OprM system is capable of efflux of irgasan, a broad-spectrum antimicrobial compound used in media selective for Pseudomonas.

Pseudomonas aeruginosa PAO4096 was induced for beta-lactamases with 6-aminopenicillanic acid. Surface changes concomitant with beta-lactamase induction were monitored. The surface hydrophobicity of the culture increased during exposure to 6-aminopenicillanic acid. The increase was associated with a change in the distribution of the O antigen in the lipopolysaccharide of treated cells. The hydrophobicity change was reversible and partially inhibited by depressed protein synthesis. The susceptibility of induced cells to rifampin was increased transiently, suggesting increased permeability of the induced cells.

The genetic basis of multiple drug resistance of Neisseria gonorrhoeae was investigated by the technique of transformation. Six different genetic loci were characterized by the type and amount of antibiotic resistance they controlled, and also by the degree of linkage to other resistance markers. A streptomycin resistance locus is linked to separate loci determining resistance to tetracycline, chloramphenicol, and erythromycin. A multiple resistance locus was identified. This genetic locus determines resistance to a variety of antibacterial agents. Lastly, a locus determining resistance to the penicillins was found which is unlinked to any other resistance locus.