Lejla Imamovic

Use of Collateral Sensitivity Networks to Design Drug Cycling Protocols That Avoid Resistance Development

Elucidation of complex collateral sensitivity networks provides rationale for new sequential drug deployment strategies that restrain antibiotic resistance development. Resistance is Futile In an emergency situation, people are often instructed to remain calm and proceed in an orderly fashion. The same advice may apply to the current antibiotic-resistance crisis. Imamovic and Sommer now show how collateral sensitivity profiles—deciphered by treating bacteria with multiple antibiotics—can help to order drug deployment in sequences that thwart the development of antibiotic resistance. Cells or organisms that have developed resistance to one drug sometimes display a greater sensitivity to a second drug often from a distinct structural class, a concept called collateral sensitivity. The authors tested whether application of this concept can aid in the management of bacterial infections by evolving resistance in Escherichia coli to 23 known antibiotics and then mapping the resulting collateral sensitivity and resistance profiles. On the basis of their derived collateral sensitivity network, the authors designed a new treatment framework—collateral sensitivity cycling—in which sequential treatment of E. coli cultures with antibiotics that display compatible collateral sensitivity profiles is predicted to select against drug-resistance development. The authors chronicled hundreds of such drug sets and validated their predictions E. coli and the antibiotics gentamicin and cefuroxime by showing that cyclical deployment of the drugs selected against resistance to either antibiotic. This proof of principle for collateral sensitivity cycling as a sustainable treatment regimen was then validated with two related bacterial pathogens. It remains to be seen whether deployment of cancer therapeutics in an orderly fashion also curbs drug resistance. New drug deployment strategies are imperative to address the problem of drug resistance, which is limiting the management of infectious diseases and cancers. We evolved resistance in Escherichia coli toward 23 drugs used clinically for treating bacterial infections and mapped the resulting collateral sensitivity and resistance profiles, revealing a complex collateral sensitivity network. On the basis of these data, we propose a new treatment framework—collateral sensitivity cycling—in which drugs with compatible collateral sensitivity profiles are used sequentially to treat infection and select against drug resistance development. We identified hundreds of such drug sets and demonstrated that the antibiotics gentamicin and cefuroxime can be deployed cyclically such that the treatment regimen selected against resistance to either drug. We then validated our findings with related bacterial pathogens. These results provide proof of principle for collateral sensitivity cycling as a sustainable treatment paradigm that may be generally applicable to infectious diseases and cancer.

Bacteriophages Carrying Antibiotic Resistance Genes in Fecal Waste from Cattle, Pigs, and Poultry

ABSTRACT This study evaluates the occurrence of bacteriophages carrying antibiotic resistance genes in animal environments. blaTEM, blaCTX-M (clusters 1 and 9), and mecA were quantified by quantitative PCR in 71 phage DNA samples from pigs, poultry, and cattle fecal wastes. Densities of 3 to 4 log10 gene copies (GC) of blaTEM, 2 to 3 log10 GC of blaCTX-M, and 1 to 3 log10 GC of mecA per milliliter or gram of sample were detected, suggesting that bacteriophages can be environmental vectors for the horizontal transfer of antibiotic resistance genes.

Quantification of Shiga Toxin-Converting Bacteriophages in Wastewater and in Fecal Samples by Real-Time Quantitative PCR

ABSTRACT Shiga toxin-converting bacteriophages (Stx phages) are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Stx phages are released from their bacterial hosts after lytic induction and remain free in the environment. Samples were analyzed for the presence of free Stx phages by an experimental approach based on the use of real-time quantitative PCR (qPCR), which enables stx to be detected in the DNA from the viral fraction of each sample. A total of 150 samples, including urban raw sewage samples, wastewater samples with fecal contamination from cattle, pigs, and poultry, and fecal samples from humans and diverse animals, were used in this study. Stx phages were detected in 70.0% of urban sewage samples (10 to 103 gene copies [GC] per ml) and in 94.0% of animal wastewater samples of several origins (10 to 1010 GC per ml). Eighty-nine percent of cattle fecal samples were positive for Stx phages (10 to 105 GC per g of sample), as were 31.8% of other fecal samples of various origins (10 to 104 GC per g of sample). The stx2 genes and stx2 variants were detected in the viral fraction of some of the samples after sequencing of stx2 fragments amplified by conventional PCR. The occurrence and abundance of Stx phages in the extraintestinal environment confirm the role of Stx phages as a reservoir of stx in the environment.

Phage-Mediated Shiga Toxin 2 Gene Transfer in Food and Water

ABSTRACT Shiga toxin (stx) transduction in various food matrices has been evaluated with lysogens of Stx phages. stx transduction events were observed for many phages under appropriate conditions. Transduction did not occur at low pH and low temperatures. A total of 103 to 104 CFU ml−1 was the minimal amount of donor and recipient strains necessary to generate transductants.

Characterizing RecA-Independent Induction of Shiga toxin2-Encoding Phages by EDTA Treatment

Background The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA. Methodology/Principal Findings The influence of EDTA on RecA-independent Stx2 phage induction was assessed, in laboratory lysogens and in EHEC strains carrying Stx2 phages in their genome, by Real-Time PCR. RecA-independent mechanisms described for phage λ induction (RcsA and DsrA) were not involved in Stx2 phage induction. In addition, mutations in the pathway for the stress response of the bacterial envelope to EDTA did not contribute to Stx2 phage induction. The effect of EDTA on Stx phage induction is due to its chelating properties, which was also confirmed by the use of citrate, another chelating agent. Our results indicate that EDTA affects Stx2 phage induction by disruption of the bacterial outer membrane due to chelation of Mg2+. In all the conditions evaluated, the pH value had a decisive role in Stx2 phage induction. Conclusions/Significance Chelating agents, such as EDTA and citrate, induce Stx phages, which raises concerns due to their frequent use in food and pharmaceutical products. This study contributes to our understanding of the phenomenon of induction and release of Stx phages as an important factor in the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) and in the emergence of new pathogenic strains.

Quantification and Evaluation of Infectivity of Shiga Toxin-Encoding Bacteriophages in Beef and Salad

ABSTRACT Stx bacteriophages in 68 samples of beef and salad were quantified by real-time quantitative PCR (qPCR). Stx phages from the samples were propagated in Escherichia coli C600, E. coli O157:H7, and Shigella strains and further quantified. Fifty percent of the samples carried infectious Stx phages that were isolated from plaques generated by lysis.

Bacteriophages and genetic mobilization in sewage and faecally polluted environments

Bacteriophages are one of the most abundant entities on the planet and are present in high concentrations within humans and animals, mostly in the gut. Phages that infect intestinal bacteria are released by defecation and remain free in extra‐intestinal environments, where they usually persist for longer than their bacterial hosts. Recent studies indicate that a large amount of the genetic information in bacterial genomes and in natural environments is of phage origin. In addition, metagenomic analysis reveals that a substantial number of bacterial genes are present in viral DNA in different environments. These facts support the belief that phages can play a significant role in horizontal gene transfer between bacteria. Bacteriophages are known to transfer genes by generalized and specialized transduction and indeed there are some examples of phages found in the environment carrying and transducing genes of bacterial origin. A successful transduction in the environment requires certain conditions, e.g. phage and bacterial numbers need to exceed certain threshold concentrations, the bacteria need to exist in an infection‐competent physiological state, and lastly, the physical conditions in the environment (pH, temperature, etc. of the supporting matrix) have to be suitable for phage infection. All three factors are reviewed here, and the available information suggests: (i) that the number of intestinal bacteria and phages in faecally contaminated environments guarantees bacteria–phage encounters, (ii) that transduction to intestinal bacteria in the environment is probable, and (iii) that transduction is more frequent than previously thought. Therefore, we suggest that phage‐mediated horizontal transfer between intestinal bacteria, or between intestinal and autochthonous bacteria in extra‐intestinal environments, might take place and that its relevance for the emergence of new bacterial strains and potential pathogens should not be ignored.

Quantification of Shiga toxin 2-encoding bacteriophages, by real-time PCR and correlation with phage infectivity.

Aims:  To evaluate a qPCR‐based protocol for the enumeration of Shiga toxin (Stx) 2 phages and to compare the results of qPCR with the number of infective Stx phage particles.

Rapid resistome mapping using nanopore sequencing

Abstract The emergence of antibiotic resistance in human pathogens has become a major threat to modern medicine. The outcome of antibiotic treatment can be affected by the composition of the gut. Accordingly, knowledge of the gut resistome composition could enable more effective and individualized treatment of bacterial infections. Yet, rapid workflows for resistome characterization are lacking. To address this challenge we developed the poreFUME workflow that deploys functional metagenomic selections and nanopore sequencing to resistome mapping. We demonstrate the approach by functionally characterizing the gut resistome of an ICU (intensive care unit) patient. The accuracy of the poreFUME pipeline is with >97% sufficient for the annotation of antibiotic resistance genes. The poreFUME pipeline provides a promising approach for efficient resistome profiling that could inform antibiotic treatment decisions in the future.

Evolution of a self-inducible cytolethal distending toxin type V-encoding bacteriophage from Escherichia coli O157:H7 to Shigella sonnei.

ABSTRACT Some cdt genes are located within the genome of inducible or cryptic bacteriophages, but there is little information about the mechanisms of cdt transfer because of the reduced number of inducible Cdt phages described. In this study, a new self-inducible Myoviridae Cdt phage (ΦAA91) was isolated from a nonclinical O157:H7 Shiga toxin-producing Escherichia coli strain and was used to lysogenize a cdt-negative strain of Shigella sonnei. We found that the phage induced from S. sonnei (ΦAA91-ss) was not identical to the original phage. ΦAA91-ss was used to infect a collection of 57 bacterial strains, was infectious in 59.6% of the strains, and was able to lysogenize 22.8% of them. The complete sequence of ΦAA91-ss showed a 33,628-bp genome with characteristics of a P2-like phage with the cdt operon located near the cosR site. We found an IS21 element composed of two open reading frames inserted within the cox gene of the phage, causing gene truncation. Truncation of cox does not affect lytic induction but could contribute to phage recombination and generation of lysogens. The IS21 element was not present in the ΦAA91 phage from E. coli, but it was incorporated into the phage genome after its transduction in Shigella. This study shows empirically the evolution of temperate bacteriophages carrying virulence genes after infecting a new host and the generation of a phage population with better lysogenic abilities that would ultimately lead to the emergence of new pathogenic strains.