Antibiotics and the ribosome

The ribosome is one of the main antibiotic targets in the cell. Recent years brought important insights into the mode of interaction of antibiotics with the ribosome and mechanisms of antibiotic action. Ribosome crystallography provided a detailed view of the interactions between antibiotics and rRNA. Advances in biochemical techniques let us better understand how the binding of small organic molecules can interfere with functions of an enzyme four orders of magnitude larger than the inhibitor. These and other achievements paved the way for the development of new ribosome-targeting antibiotics, some of which have already entered medical practice. The recent progress, problems and new directions of research of ribosome-targeting antibiotics are discussed in this review.     

Genetic Architecture of Intrinsic Antibiotic Susceptibility

Background

Antibiotic exposure rapidly selects for more resistant bacterial strains, and both a drug''s chemical structure and a bacterium''s cellular network affect the types of mutations acquired.

Methodology/Principal Findings

To better characterize the genetic determinants of antibiotic susceptibility, we exposed a transposon-mutagenized library of Escherichia coli to each of 17 antibiotics that encompass a wide range of drug classes and mechanisms of action. Propagating the library for multiple generations with drug concentrations that moderately inhibited the growth of the isogenic parental strain caused the abundance of strains with even minor fitness advantages or disadvantages to change measurably and reproducibly. Using a microarray-based genetic footprinting strategy, we then determined the quantitative contribution of each gene to E. coli''s intrinsic antibiotic susceptibility. We found both loci whose removal increased general antibiotic tolerance as well as pathways whose down-regulation increased tolerance to specific drugs and drug classes. The beneficial mutations identified span multiple pathways, and we identified pairs of mutations that individually provide only minor decreases in antibiotic susceptibility but that combine to provide higher tolerance.

Conclusions/Significance

Our results illustrate that a wide-range of mutations can modulate the activity of many cellular resistance processes and demonstrate that E. coli has a large mutational target size for increasing antibiotic tolerance. Furthermore, the work suggests that clinical levels of antibiotic resistance might develop through the sequential accumulation of chromosomal mutations of small individual effect.     

Monitoring antibacterial permeabilization in real time using time-resolved flow cytometry

Despite the intensive study of antibiotic-induced bacterial permeabilization, its kinetics and molecular mechanism remain largely elusive. A new methodology that extends the concept of the live–dead assay in flow cytometry to real time-resolved detection was used to overcome these limitations. The antimicrobial activity of pepR was monitored in time-resolved flow cytometry for three bacterial strains: Escherichia coli (ATCC 25922), E. coli K-12 (CGSC Strain 4401) and E. coli JW3596-1 (CGSC Strain 11805). The latter strain has truncated lipopolysaccharides (LPS) in the outer membrane. This new methodology provided information on the efficacy of the antibiotics and sheds light on their mode of action at membrane-level. Kinetic data regarding antibiotic binding and lytic action were retrieved. Membrane interaction and permeabilization events differ significantly among strains. The truncation of LPS moieties does not hamper AMP binding but compromises membrane disruption and bacterial killing. We demonstrated the usefulness of time-resolved flow cytometry to study antimicrobial-induced permeabilization by collecting kinetic data that contribute to characterize the action of antibiotics directly on bacteria.     

Inter-species interactions alter antibiotic efficacy in bacterial communities

The efficacy of antibiotic treatments targeting polymicrobial communities is not well predicted by conventional in vitro susceptibility testing based on determining minimum inhibitory concentration (MIC) in monocultures. One reason for this is that inter-species interactions can alter the community members’ susceptibility to antibiotics. Here we quantify, and identify mechanisms for, community-modulated changes of efficacy for clinically relevant antibiotics against the pathogen Pseudomonas aeruginosa in model cystic fibrosis (CF) lung communities derived from clinical samples. We demonstrate that multi-drug resistant Stenotrophomonas maltophilia can provide high levels of antibiotic protection to otherwise sensitive P. aeruginosa. Exposure protection to imipenem was provided by chromosomally encoded metallo-β-lactamase that detoxified the environment; protection was dependent upon S. maltophilia cell density and was provided by S. maltophilia strains isolated from CF sputum, increasing the MIC of P. aeruginosa by up to 16-fold. In contrast, the presence of S. maltophilia provided no protection against meropenem, another routinely used carbapenem. Mathematical ordinary differential equation modelling shows that the level of exposure protection provided against different carbapenems can be explained by differences in antibiotic efficacy and inactivation rate. Together, these findings reveal that exploitation of pre-occurring antimicrobial resistance, and inter-specific competition, can have large impacts on pathogen antibiotic susceptibility, highlighting the importance of microbial ecology for designing successful antibiotic treatments for multispecies communities.Subject terms: Antibiotics, Bacterial infection, Microbial ecology     

Minimal biophysical model of combined antibiotic action

Phenomenological relations such as Ohm’s or Fourier’s law have a venerable history in physics but are still scarce in biology. This situation restrains predictive theory. Here, we build on bacterial “growth laws,” which capture physiological feedback between translation and cell growth, to construct a minimal biophysical model for the combined action of ribosome-targeting antibiotics. Our model predicts drug interactions like antagonism or synergy solely from responses to individual drugs. We provide analytical results for limiting cases, which agree well with numerical results. We systematically refine the model by including direct physical interactions of different antibiotics on the ribosome. In a limiting case, our model provides a mechanistic underpinning for recent predictions of higher-order interactions that were derived using entropy maximization. We further refine the model to include the effects of antibiotics that mimic starvation and the presence of resistance genes. We describe the impact of a starvation-mimicking antibiotic on drug interactions analytically and verify it experimentally. Our extended model suggests a change in the type of drug interaction that depends on the strength of resistance, which challenges established rescaling paradigms. We experimentally show that the presence of unregulated resistance genes can lead to altered drug interaction, which agrees with the prediction of the model. While minimal, the model is readily adaptable and opens the door to predicting interactions of second and higher-order in a broad range of biological systems.     

Comparative genome analysis and characterization of a MDR Klebsiella variicola

Klebsiella variicola is an emerging pathogen responsible for causing blood-stream infections, urinary and respiratory tract related diseases in humans. In this report, we describe the genome sequence data and phenotypic characterization of K. variicola strain KV093 isolated from India. Comparative genome sequence analysis revealed the presence of genes linked with virulence, iron acquisition and transport, type 1 and type 3 pili, secretion systems including the capsular gene cluster. The plant-associated genes such as nitrogen fixation, growth and defense mechanisms against oxidative stress were also identified. On performing antibiotic susceptibility testing, growth inhibition, and stress challenge assays it was observed that the drug resistant K. variicola KV093 exhibited cross resistance to various antibiotics, antiseptics, including disinfectants. This report highlights the arsenal of virulence and antibiotic resistance determinants in K. variicola KV093, an effort emphasizing the current pressing need for regular surveillance of K. variicola strains especially in India.     

Influence of Electromagnetic Signal of Antibiotics Excited by Low-Frequency Pulsed Electromagnetic Fields on Growth of Escherichia coli

Energy medicine (EM) provides a new medical choice for patients, and its advantages are the noninvasive detection and nondrug treatment. An electromagnetic signal, a kind of EM, induced from antibiotic coupling with weak, extremely low-frequency pulsed electromagnetic fields (PEMFs) is utilized for investigating the growth speed of Escherichia coli (E. coli). PEMFs are produced by solenoidal coils for coupling the electromagnetic signal of antibiotics (penicillin). The growth retardation rate (GRR) of E. coli is used to investigate the efficacy of the electromagnetic signal of antibiotics. The E. coli is cultivated in the exposure of PEMFs coupling with the electromagnetic signal of antibiotics. The maximum GRR of PEMFs with and without the electromagnetic signal of antibiotics on the growth of E. coli cells in the logarithmic is 17.4 and 9.08 %, respectively. The electromagnetic signal of antibiotics is successfully coupled by the electromagnetic signal coupling instrument to affect the growth of E. coli. In addition, the retardation effect on E. coli growth can be improved of by changing the carrier frequency of PEMFs coupling with the electromagnetic signal of antibiotics. GRR caused by the electromagnetic signal of antibiotics can be fixed by a different carrier frequency in a different phase of E. coli growth.     

Antibiotic growth promoter olaquindox increases pathogen susceptibility in fish by inducing gut microbiota dysbiosis

Low dose antibiotics have been used as growth promoters in livestock and fish. The use of antibiotics has been associated with reduced pathogen infections in livestock. In contrast, antibiotic growth promoter has been suspected of leading to disease outbreaks in aquaculture. However, this phenomenon is circumstantial and has not been confirmed in experimental conditions. In this study,we showed that antibiotic olaquindox increased the susceptibility of zebrafish to A. hydrophila infection. Olaquindox led to profound alterations in the intestinal microbiota of zebrafish, with a drastic bloom of Enterobacter and diminishing of Cetobacterium. Moreover, the innate immune responses of zebrafish were compromised by olaquindox(P0.05). Transfer of microbiota to GF zebrafish indicated that while the immuo-suppression effect of olaquindox is a combined effect mediated by both OLA-altered micro biota and direct action of the antibiotic(P0.05), the increased pathogen susceptibility was driven by the OLA-altered microbiota and was not dependent on direct antibiotic effect. Taken together, these data indicate that low level of OLA induced gut microbiota dysbiosis in zebrafish, which led to increased pathogen susceptibility.     

Using a Sequential Regimen to Eliminate Bacteria at Sublethal Antibiotic Dosages

We need to find ways of enhancing the potency of existing antibiotics, and, with this in mind, we begin with an unusual question: how low can antibiotic dosages be and yet bacterial clearance still be observed? Seeking to optimise the simultaneous use of two antibiotics, we use the minimal dose at which clearance is observed in an in vitro experimental model of antibiotic treatment as a criterion to distinguish the best and worst treatments of a bacterium, Escherichia coli. Our aim is to compare a combination treatment consisting of two synergistic antibiotics to so-called sequential treatments in which the choice of antibiotic to administer can change with each round of treatment. Using mathematical predictions validated by the E. coli treatment model, we show that clearance of the bacterium can be achieved using sequential treatments at antibiotic dosages so low that the equivalent two-drug combination treatments are ineffective. Seeking to treat the bacterium in testing circumstances, we purposefully study an E. coli strain that has a multidrug pump encoded in its chromosome that effluxes both antibiotics. Genomic amplifications that increase the number of pumps expressed per cell can cause the failure of high-dose combination treatments, yet, as we show, sequentially treated populations can still collapse. However, dual resistance due to the pump means that the antibiotics must be carefully deployed and not all sublethal sequential treatments succeed. A screen of 136 96-h-long sequential treatments determined five of these that could clear the bacterium at sublethal dosages in all replicate populations, even though none had done so by 24 h. These successes can be attributed to a collateral sensitivity whereby cross-resistance due to the duplicated pump proves insufficient to stop a reduction in E. coli growth rate following drug exchanges, a reduction that proves large enough for appropriately chosen drug switches to clear the bacterium.     

Prevalence of Veterinary Antibiotics and Antibiotic-Resistant Escherichia coli in the Surface Water of a Livestock Production Region in Northern China

This study investigated the occurrence of 12 veterinary antibiotics (VAs) and the susceptibility of Escherichia coli (E. coli) in a rural water system that was affected by livestock production in northern China. Each of the surveyed sites was determined with at least eight antibiotics with maximum concentration of up to 450 ng L⁻¹. The use of VAs in livestock farming probably was a primary source of antibiotics in the rivers. Increasing total antibiotics were measured from up- to mid- and downstream in the two tributaries. Eighty-eight percent of the 218 E. coli isolates that were derived from the study area exhibited, in total, 48 resistance profiles against the eight examined drugs. Significant correlations were found among the resistance rates of sulfamethoxazole-trimethoprim, chloromycetin and ampicillin as well as between tetracycline and chlortetracycline, suggesting a possible cross-selection for resistance among these drugs. The E. coli resistance frequency also increased from up- to midstream in the three rivers. E. coli isolates from different water systems showed varying drug numbers of resistance. No clear relationship was observed in the antibiotic resistance frequency with corresponding antibiotic concentration, indicating that the antibiotic resistance for E. coli in the aquatic environment might be affected by factors besides antibiotics. High numbers of resistant E. coli were also isolated from the conserved reservoir. These results suggest that rural surface water may become a large pool of VAs and resistant bacteria. This study contributes to current information on VAs and resistant bacteria contamination in aquatic environments particularly in areas under intensive agriculture. Moreover, this study indicates an urgent need to monitor the use of VAs in animal production, and to control the release of animal-originated antibiotics into the environment.     

A multi-type branching model with varying environment for bacterial dynamics with postantibiotic effect

A multi-type branching process with varying environment was used to construct a pharmacokinetic/pharmacodynamic (PK/PD) model that captures the postantibiotic effect (PAE) seen in bacterial populations after exposure of antibiotics. This phenomenon of continued inhibition of bacterial growth even after removal of the antibiotic from the growth medium is of high relevance in the context of optimizing dosing regimens. The clinical implication of long PAEs lies in the interesting possibility of increasing the intervals between drug administrations.The model structure is generalizable to most types of antibiotics and is useful both as a theoretical framework for understanding the time properties of PAE and to explore optimal antibiotic dosing regimens. Data from an in vitro study with Escherichia coli exposed to different dosing regimens of cefotaxime were used to evaluate the model.     

P1 Ref Endonuclease: A Molecular Mechanism for Phage-Enhanced Antibiotic Lethality

Ref is an HNH superfamily endonuclease that only cleaves DNA to which RecA protein is bound. The enigmatic physiological function of this unusual enzyme is defined here. Lysogenization by bacteriophage P1 renders E. coli more sensitive to the DNA-damaging antibiotic ciprofloxacin, an example of a phenomenon termed phage-antibiotic synergy (PAS). The complementary effect of phage P1 is uniquely traced to the P1-encoded gene ref. Ref is a P1 function that amplifies the lytic cycle under conditions when the bacterial SOS response is induced due to DNA damage. The effect of Ref is multifaceted. DNA binding by Ref interferes with normal DNA metabolism, and the nuclease activity of Ref enhances genome degradation. Ref also inhibits cell division independently of the SOS response. Ref gene expression is toxic to E. coli in the absence of other P1 functions, both alone and in combination with antibiotics. The RecA proteins of human pathogens Neisseria gonorrhoeae and Staphylococcus aureus serve as cofactors for Ref-mediated DNA cleavage. Ref is especially toxic during the bacterial SOS response and the limited growth of stationary phase cultures, targeting aspects of bacterial physiology that are closely associated with the development of bacterial pathogen persistence.     

PA2800 Plays an Important Role in Both Antibiotic Susceptibility and Virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important human pathogen which causes a variety of infections. P. aeruginosa infections are often difficult to treat due to the pathogen’s resistance to many antibiotics. Previously, it has been reported that a transposon insertion mutant in gene PA2800 of P. aeruginosa PAO1 was more sensitive to tetracycline and ciprofloxacin. Further characterization of this gene, a vacJ homolog, in this study indicated that this gene plays an important role in both antibiotic susceptibility and virulence in P. aeruginosa. The role of PA2800 in antibiotic susceptibility probably signifies its involvement in maintaining outer membrane stability, similar to the role of vacJ in E. coli and Shigella flexneri. However, in contrast to vacJ in other bacteria, PA2800 also affects antibiotic susceptibility by affecting the expression of oprH in P. aeruginosa. As shown by in vivo studies using a Drosophila melanogaster infection model, significantly increased virulence was observed in the PA2800 mutant when compared to the wild type, and such a difference is likely a result of disrupted outer membrane stability and altered expression of znuA in the mutant. The role of PA2800 or vacJ in antibiotic susceptibility and pathogenicity seems to be unique in P. aeruginosa in which it affects both outer membrane stability as well as gene expression.     

Systematic discovery of drug interaction mechanisms

Drug combinations are increasingly important in disease treatments, for combating drug resistance, and for elucidating fundamental relationships in cell physiology. When drugs are combined, their individual effects on cells may be amplified or weakened. Such drug interactions are crucial for treatment efficacy, but their underlying mechanisms remain largely unknown. To uncover the causes of drug interactions, we developed a systematic approach based on precise quantification of the individual and joint effects of antibiotics on growth of genome‐wide Escherichia coli gene deletion strains. We found that drug interactions between antibiotics representing the main modes of action are highly robust to genetic perturbation. This robustness is encapsulated in a general principle of bacterial growth, which enables the quantitative prediction of mutant growth rates under drug combinations. Rare violations of this principle exposed recurring cellular functions controlling drug interactions. In particular, we found that polysaccharide and ATP synthesis control multiple drug interactions with previously unexplained mechanisms, and small molecule adjuvants targeting these functions synthetically reshape drug interactions in predictable ways. These results provide a new conceptual framework for the design of multidrug combinations and suggest that there are universal mechanisms at the heart of most drug interactions.     

Lipopolysaccharide as an antibiotic target

Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii are amongst the highest priority drug-resistant pathogens, for which new antibiotics are urgently needed. Whilst antibiotic drug development is inherently challenging, this is particularly true for Gram-negative bacteria due to the presence of the outer membrane, a highly selective permeability barrier that prevents the ingress of several classes of antibiotic. This selectivity is largely due to an outer leaflet composed of the glycolipid lipopolysaccharide (LPS), which is essential for the viability of almost all Gram-negative bacteria. This essentiality, coupled with the conservation of the synthetic pathway across species and recent breakthroughs in our understanding of transport and membrane homeostasis has made LPS an attractive target for novel antibiotic drug development. Several different targets have been explored and small molecules developed that show promising activity in vitro. However, these endeavours have met limited success in clinical testing and the polymyxins, discovered more than 70 years ago, remain the only LPS-targeting drugs to enter the clinic thus far. In this review, we will discuss efforts to develop therapeutic inhibitors of LPS synthesis and transport and the reasons for limited success, and explore new developments in understanding polymyxin mode of action and the identification of new analogues with reduced toxicity and enhanced activity.     

Homology modeling of phosphoryl thymidine kinase of enterohemorrhagic Escherichia coli OH: 157

Enterohemorrhagic Escherichia coli (EHEC) are source of emerging infectious disease in India. Escherichia coli O157:H7 is an EHEC strain showing multiple antibiotic resistances and the cause of infantile diarrhea and hemolytic uremic syndrome worldwide. A novel strategy to counteract multiple antibiotic resistant organisms is to design drugs which specifically target metabolic pathways such as thiamine biosynthetic pathways found exclusively in prokaryotes. Homology modeling was used for model building of a terminal thiamine biosynthesis enzyme phosphoryl thymidine kinase (Thi E) using Geno3D, Swiss Model and Modeller. The best model was selected based on overall stereochemical quality. The potential ligand binding sites in the model were identified by CASTp server. The validated theoretical model of the 3D structure of the thiE protein of E. coli O157:H7 was predicted using a thiamine phosphate pyrophosphatase from Pyrococcus furiosus (PDB ID: 1X13_A) as template. The active pockets of ligand binding sites in the enzyme were identified. In this study, phosphoryl thymidine kinase (thi E), a terminal enzyme in the thiamine biosynthesis pathway in the pathogen has been modeled to be used in future as a potential drug target by the design of suitable inhibitors.     

圈养猕猴志贺氏菌的分离鉴定及药敏分析

目的确定猕猴感染志贺氏菌的状况,寻找有效治疗措施。方法采用不同选择培养基对13份病猴粪便样品进行分离培养、细菌革兰氏染色、镜检,并对分离的疑似菌株进行细菌生化鉴定和分子鉴定,经小白鼠致病性实验后,再用纸片扩散法测定分离菌株对23种抗生素的敏感性。结果从13份猕猴粪便样品中共检出12株志贺氏菌,检出率为92.31%,其中痢疾志贺菌(A群)1株、福氏志贺菌(B群)10株、宋内氏志贺菌(D群)1株;致病性试验结果表明,12株菌均能在72h内致死小白鼠,并能回收到注射的菌株;药敏试验结果表明,本实验中分离到的志贺氏菌对头孢噻肟(86.49%)最敏感,对头孢三嗪(75.00%)、头孢他啶(66.67%)次之,对多粘菌素B、羧苄西林、苄唑西林素等抗生素耐药性强。结论初步确定猕猴感染志贺氏菌普遍存在,进而引起腹泻、痢疾的可能性较大,头孢噻肟等为最敏感药物。     

Rapid Biosensor for Detection of Antibiotic-Selective Growth of Escherichia coli

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated in common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The calculated mass sensitivity according to the mechanical properties of the cantilever sensor is ~50 pg/Hz; this mass corresponds to an approximate sensitivity of ~100 Escherichia coli cells. The sensor is able to detect active growth of E. coli cells within 1 h. The starting number of E. coli cells initially attached to the sensor cantilever was, on average, ~1,000 cells. Furthermore, this method allows the detection of selective growth of E. coli within only 2 h by adding antibiotics to the nutritive layers. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of selective bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing.     

Increased Bacterial Hemolytic Activity is Conferred by Expression of TlyA Methyltransferase but not by its 2′-O-methylation of the Ribosome

Bacterial 2′-O-methyltransferase TlyA methylates either both nucleotide C1409 of 16S rRNA and C1920 of 23S rRNA or only the C1920. Both ribosomal methylations increase bacterial susceptibility to ribosome-targeting antibiotics capreomycin and viomycin. However, TlyA has been suggested to also function as a hemolysin. Here, heterologous expression of TlyA from six diverse bacteria (including Mycobacterium tuberculosis and M. smegmatis) was found to increase hemolytic ability in the Escherichia coli host. Characterizing E. coli strains expressing mycobacterial TlyA with mutated rRNA recognition domain and impaired rRNA methylations showed that the abolished C1409 methylation altogether with significantly reduced C1920 methylation did not affect E. coli hemolytic activity. Thus, the increased bacterial hemolytic function is not likely a consequence of TlyA-mediated methylations of the ribosome. Purified water-soluble TlyA showed a weak concentration-dependent hemolysis in vitro. Therefore, the TlyA isoform alone is not a potent hemolysin. The results suggested that the bacterial hemolytic function might relate to the over-expression of TlyA and its interaction to other non-ribosomal target that is associated with the hemolytic ability.     

A new look at the drug-resistance investigation of uropathogenic <Emphasis Type="Italic">E. coli</Emphasis> strains

Bacterial drug resistance and uropathogenic tract infections are among the most important issues of current medicine. Uropathogenic Escherichia coli strains are the primary factor of this issue. This article is the continuation of the previous study, where we used Kohonen relations to predict the direction of drug resistance. The characterized collection of uropathogenic E. coli strains was used for microbiological (the disc diffusion method for antimicrobial susceptibility testing), chemical (ATR/FT-IR) and mathematical (artificial neural networks, Ward’s hierarchical clustering method, the analysis of distributions of inhibition zone diameters for antibiotics, Cohen’s kappa measure of agreement) analysis. This study presents other potential tools for the epidemiological differentiation of E. coli strains. It is noteworthy that ATR/FT-IR technique has turned out to be useful for the quick and simple identification of MDR strains. Also, diameter zones of resistance of this E. coli population were compared to the population of E. coli strains published by EUCAST. We observed the bacterial behaviors toward particular antibiotics in comparison to EUCAST bacterial collections. Additionally, we used Cohen’s kappa to show which antibiotics from the same class are closely related to each other and which are not. The presented associations between antibiotics may be helpful in selecting the proper therapy directions. Here we present an adaptation of interdisciplinary studies of drug resistance of E. coli strains for epidemiological and clinical investigations. The obtained results may be some indication in deciding on antibiotic therapy.