Beta-lactamases and bacterial resistance to antibiotics

The efficiency of β-lactam antibiotics, which are among our most useful chemotherapeutic weapons, is continuously challenged by the emergence of resistant bacterial strains. This is most often due to the production of β-lactamases by the resistant cells. These enzymes inactivate the antibiotics by hydrolysing the β-lactam amide bond. The elucidation of the structures of some β-lactamases by X-ray crystallography has provided precious insights into their catalytic mechanisms and revealed unsuspected similarities with the DD-transpeptidases, the bacterial enzymes which constitute the lethal targets of β-lactams. Despite numerous kinetic, structural and site-directed mutagenesis studies, we have not completely succeeded in explaining the diversity of the specificity profiles of β-lactamases and their surprising catalytic power. The solutions to these problems represent the cornerstones on which better antibiotics can be designed, hopefully on a rational basis.     

Reducing bacterial resistance to antibiotics with ultrasound

The effect of erythromycin on planktonic cultures of Psedomonas aeruginosa, with and without application of 70 kHz ultrasound, was studied. Ultrasound was applied at levels that had no inhibitory effect on cultures of Ps. aeruginosa. Ultrasound in combination with erythromycin reduced the viability of Ps. aeruginosa by 1-2 orders of magnitude compared with antibiotic alone, even at concentrations below the minimum inhibitory concentration (MIC). Electron-spin resonance studies suggest that ultrasound induces uptake of antibiotic by perturbing or stressing the membrane. This application of ultrasound may be useful for expanding the number of drugs available for treating localized infections by rendering bacteria susceptible to normally ineffective antibiotics.     

Persistence and resistance as complementary bacterial adaptations to antibiotics

Bacterial persistence represents a simple of phenotypic heterogeneity, whereby a proportion of cells in an isogenic bacterial population can survive exposure to lethal stresses such as antibiotics. In contrast, genetically based antibiotic resistance allows for continued growth in the presence of antibiotics. It is unclear, however, whether resistance and persistence are complementary or alternative evolutionary adaptations to antibiotics. Here, we investigate the co‐evolution of resistance and persistence across the genus Pseudomonas using comparative methods that correct for phylogenetic nonindependence. We find that strains of Pseudomonas vary extensively in both their intrinsic resistance to antibiotics (ciprofloxacin and rifampicin) and persistence following exposure to these antibiotics. Crucially, we find that persistence correlates positively to antibiotic resistance across strains. However, we find that different genes control resistance and persistence implying that they are independent traits. Specifically, we find that the number of type II toxin–antitoxin systems (TAs) in the genome of a strain is correlated to persistence, but not resistance. Our study shows that persistence and antibiotic resistance are complementary, but independent, evolutionary adaptations to stress and it highlights the key role played by TAs in the evolution of persistence.     

Cysteine metabolism-related genes and bacterial resistance to potassium tellurite

Tellurite exerts a deleterious effect on a number of small molecules containing sulfur moieties that have a recognized role in cellular oxidative stress. Because cysteine is involved in the biosynthesis of glutathione and other sulfur-containing compounds, we investigated the expression of Geobacillus stearothermophilus V cysteine-related genes cobA, cysK, and iscS and Escherichia coli cysteine regulon genes under conditions that included the addition of K₂TeO₃ to the culture medium. Results showed that cell tolerance to tellurite correlates with the expression level of the cysteine metabolic genes and that these genes are up-regulated when tellurite is present in the growth medium.     

Evolution of bacterial resistance to antibiotics during the last three decades.

Bacterial resistance to antibiotics is often plasmid-mediated and the associated genes encoded by transposable elements. These elements play a central role in evolution by providing mechanisms for the generation of diversity and, in conjunction with DNA transfer systems, for the dissemination of resistances to other bacteria. At the University Hospital of Zaragoza, extensive efforts have been made to define both the dissemination and evolution of antibiotic resistance by studying the transferable R plasmids and transposable elements. Here we describe the research on bacterial resistance to antibiotics in which many authors listed in the references have participated. The aspects of bacterial resistance dealt with are: (i) transferable resistance mediated by R plasmids in Gram-negative bacteria, (ii) R plasmid-mediated resistance to apramycin and hygromycin in clinical strains, (iii) the transposon Tn1696 and the integron In4, (iv) expression of Escherichia coli resistance genes in Haemophilus influenzae, (v) aminoglycoside-modifying-enzymes in the genus Mycobacterium with no relation to resistance, and (vi) macrolide-resistance and new mechanisms developed by Gram-positive bacteria.     

Mechanisms of resistance to antibiotics

Microbial resistance to antibiotics is manifested by changes in antibiotic permeability, alteration of target molecules, enzymatic degradation of the antibiotics, and efflux of antimicrobials from the cytosol. Bacteria and other microorganisms use all of these mechanisms to evade the toxic effects of antibiotics. Recent research on the molecular aspects of these mechanisms, often informed by atomic resolution structures of proteins, enzymes and nucleic acids involved in these processes, has deepened our understanding of antibiotic action and resistance and, in several cases, spurred the development of strategies to overcome resistance in vitro and in vivo.     

Chloraminated drinking water does not generate bacterial resistance to antibiotics in Pseudomonas aeruginosa biofilms

Aim: To determine if exposure of Pseudomonas aeruginosa biofilms to chloraminated drinking water can lead to individual bacteria with resistance to antibiotics. Methods and Results: Biofilms of P. aeruginosa PA14 were grown in drinking water in a Kadouri drip‐fed reactor; the biofilms were treated with either 0·5 mg l^‐1 or 1·0 mg l^‐1 of chloramine for 15 or 21 days; control biofilms were grown in water without chloramine. Fewer isolates with antibiotic resistance were obtained from the chloramine‐treated biofilms as compared to the control. Minimum inhibitory concentrations (MIC) for selected antibiotic‐resistant isolates were determined using ciprofloxacin, tobramycin, gentamicin, rifampicin and chloramphenicol. All of the isolates tested had increased resistance over the wildtype to ciprofloxacin, rifampicin and chloramphenicol, but were not resistant to tobramycin or gentamicin. Conclusions: Under these test conditions, there was no detectable increase in antibiotic resistance in P. aeruginosa exposed as biofilms to disinfectant residues in chloraminated drinking water. Significance and Impact of the study: Chloramine in drinking water, while unable to kill biofilm bacteria, does not increase the potential of P. aeruginosa to become resistant to antibiotics.     

The bacterial phleomycin resistance geneble as a dominant selectable marker inChlamydomonas

A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5′ and 3′ untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg⁺ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (Pm^R) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the Pm^R transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a Pm^R transformant to wild-type strains. Direct selection of Pm^R transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.     

Growth-dependent bacterial susceptibility to ribosome-targeting antibiotics

Bacterial growth environment strongly influences the efficacy of antibiotic treatment, with slow growth often being associated with decreased susceptibility. Yet in many cases, the connection between antibiotic susceptibility and pathogen physiology remains unclear. We show that for ribosome-targeting antibiotics acting on Escherichia coli, a complex interplay exists between physiology and antibiotic action; for some antibiotics within this class, faster growth indeed increases susceptibility, but for other antibiotics, the opposite is true. Remarkably, these observations can be explained by a simple mathematical model that combines drug transport and binding with physiological constraints. Our model reveals that growth-dependent susceptibility is controlled by a single parameter characterizing the ‘reversibility’ of ribosome-targeting antibiotic transport and binding. This parameter provides a spectrum classification of antibiotic growth-dependent efficacy that appears to correspond at its extremes to existing binary classification schemes. In these limits, the model predicts universal, parameter-free limiting forms for growth inhibition curves. The model also leads to non-trivial predictions for the drug susceptibility of a translation mutant strain of E. coli, which we verify experimentally. Drug action and bacterial metabolism are mechanistically complex; nevertheless, this study illustrates how coarse-grained models can be used to integrate pathogen physiology into drug design and treatment strategies.