Synergy between Mecillinam and Ceftazidime/Avibactam or Avibactam against Multi-Drug-Resistant Carbapenemase-Producing Escherichia coli and Klebsiella pneumoniae.

Background: Carbapenemase-producing Klebsiella pneumoniae and Escherichia coli have become a significant global health challenge. This has created an urgent need for new treatment modalities. We evaluated the efficacy of mecillinam in combination with either avibactam or ceftazidime/avibactam against carbapenemase-producing clinical isolates. Materials and methods: Nineteen MDR clinical isolates of K. pneumoniae and E. coli were selected for the presence of blaKPC, blaNDM, blaOXA or blaIMP based on whole-genome sequencing and phenotypic susceptibility testing. We tested the synergy between mecillinam and avibactam or ceftazidime/avibactam. We used time−kill studies in vitro and a mouse peritonitis/sepsis model to confirm the synergistic effect. We investigated avibactam's impact on mecillinam´s affinity for penicillin-binding proteins with a Bocillin assay, and cell changes with phase-contrast and confocal laser scanning microscopy. Results: Mecillinam combined with ceftazidime/avibactam or avibactam substantially reduced MICs (from up to >256 µg/mL to <0.0016 µg/mL) for 17/18 strains. Significant log-CFU reductions were confirmed in time−kill and in vivo experiments. The Bocillin assay did not reveal changes. Conclusion: Mecillinam in combination with avibactam or ceftazidime/avibactam has a notable effect on most types of CPEs, both in vitro and in vivo. The mecillinam/avibactam combination treatment could be a new efficient antibiotic treatment against multi-drug-resistant carbapenemase-producing Gram-negative pathogens.

Activating the Cpx response induces tolerance to antisense PNA delivered by an arginine-rich peptide in Escherichia coli.

Cell-penetrating peptides (CPPs) are increasingly used for cellular drug delivery in both pro- and eukaryotic cells, and oligoarginines have attracted special attention. How arginine-rich CPPs translocate across the cell envelope, particularly for prokaryotes, is still unknown. Arginine-rich CPPs efficiently deliver antimicrobial peptide nucleic acid (PNA) to its intracellular mRNA target in bacteria. We show that resistance to PNA conjugated to an arginine-rich CPP in Escherichia coli requires multiple genetic modifications and is specific for the CPP part and not to the PNA part. An integral part of the resistance was the constitutively activated Cpx-envelope stress response system (cpx∗), which decreased the cytoplasmic membrane potential. This indicates an indirect energy-dependent uptake mechanism for antimicrobials conjugated to arginine-rich CPPs. In agreement, cpx∗ mutants showed low-level resistance to aminoglycosides and an arginine-rich CPP conjugated to a peptide targeting the DNA sliding clamp, i.e., similar uptake in E. coli for these antimicrobial compounds.

Arresting chromosome replication upon energy starvation in Escherichia coli.

Most organisms possess several cell cycle checkpoints to preserve genome stability in periods of stress. Upon starvation, the absence of chromosomal duplication in the bacterium Escherichia coli is ensured by holding off commencement of replication. During normal growth, accumulation of the initiator protein DnaA along with cell cycle changes in its activity, ensure that DNA replication starts only once per cell cycle. Upon nutrient starvation, the prevailing model is that an arrest in DnaA protein synthesis is responsible for the absence of initiation. Recent indications now suggest that DnaA degradation may also play a role. Here we comment on the implications of this potential new layer of regulation.

Antisense inhibition of the Escherichia coli NrdAB aerobic ribonucleotide reductase is bactericidal due to induction of DNA strand breaks.

BACKGROUND: Antisense peptide nucleic acids (PNAs) constitute an alternative to traditional antibiotics, by their ability to silence essential genes. OBJECTIVES: To evaluate the antibacterial effects of antisense PNA-peptide conjugates that target the gene encoding the alpha subunit (NrdA) of the Escherichia coli ribonucleotide reductase (RNR). METHODS: Bacterial susceptibility of a series of NrdA-targeting PNAs was studied by MIC determination and time-kill analysis. Western-blot analysis, gene complementation and synergy with hydroxyurea were employed to determine the efficiency of NrdA-PNA antisense treatment. The effect on chromosome replication was addressed by determining the DNA synthesis rate, by flow cytometry analysis, by quantitative PCR and by fluorescence microscopy. The use of DNA repair mutants provided insight into the bactericidal action of NrdA-PNA. RESULTS: Treatment with NrdA-PNA specifically inhibited growth of E. coli, as well as NrdA protein translation at 4 µM. Also, the DNA synthesis rate was reduced, preventing completion of chromosome replication and resulting in formation of double-stranded DNA breaks and cell death. CONCLUSIONS: These data present subunits of the NrdAB RNR as a target for future antisense microbial agents and provide insight into the bacterial physiological response to RNR-targeting antimicrobials.

Energy Starvation Induces a Cell Cycle Arrest in Escherichia coli by Triggering Degradation of the DnaA Initiator Protein.

During steady-state Escherichia coli growth, the amount and activity of the initiator protein, DnaA, controls chromosome replication tightly so that initiation only takes place once per origin in each cell cycle, regardless of growth conditions. However, little is known about the mechanisms involved during transitions from one environmental condition to another or during starvation stress. ATP depletion is one of the consequences of long-term carbon starvation. Here we show that DnaA is degraded in ATP-depleted cells. A chromosome replication initiation block is apparent in such cells as no new rounds of DNA replication are initiated while replication events that have already started proceed to completion.

Counting Replication Origins to Measure Growth of Pathogens.

For the past several decades, the success of bacterial strains in infecting their host has been essentially ascribed to the presence of canonical virulence genes. While it is unclear how much growth rate impacts the outcome of an infection, it is long known that the efficacy of the most commonly used antibiotics is correlated to growth. This applies especially to -lactams, whose efficacy is nearly abolished when cells grow very slowly. It is therefore reasonable to assume that a niche or genetic dependent change in growth rate could contribute to the variability in the outcome of antibiotic therapy. However, little is known about the growth rate of pathogens or their pathotypes in their host.

Short-term kinetics of rRNA degradation in Escherichia coli upon starvation for carbon, amino acid or phosphate.

Ribosomes are absolutely essential for growth but are, moreover, energetically costly to produce. Therefore, it is important to adjust the cellular ribosome levels according to the environmental conditions in order to obtain the highest possible growth rate while avoiding energy wastage on excess ribosome biosynthesis. Here we show, by three different methods, that the ribosomal RNA content of Escherichia coli is downregulated within minutes of the removal of an essential nutrient from the growth medium, or after transcription initiation is inhibited. The kinetics of the ribosomal RNA reduction vary depending on which nutrient the cells are starved for. The number of ribosomes per OD unit of cells is roughly halved after 80 min of starvation for isoleucine or phosphate, while the ribosome reduction is less extensive when the cells are starved for glucose. Collectively, the results presented here support the simple model proposed previously, which identifies the inactive ribosomal subunits as the substrates for degradation, since the most substantial rRNA degradation is observed under the starvation conditions that most directly affect the protein synthesis.

Growth Rate of Escherichia coli During Human Urinary Tract Infection: Implications for Antibiotic Effect.

Escherichia coli is the primary cause of urinary tract infection (UTI), which is one of the most frequent human infections. While much is understood about the virulence factors utilized by uropathogenic E. coli (UPEC), less is known about the bacterial growth dynamics taking place during infection. Bacterial growth is considered essential for successful host colonization and infection, and most antibiotics in clinical use depend on active bacterial growth to exert their effect. However, a means to measure the in situ bacterial growth rate during infection has been lacking. Due to faithful coordination between chromosome replication and cell growth and division in E. coli, chromosome replication provides a quantitative measure of the bacterial growth rate. In this study, we explored the potential for inferring in situ bacterial growth rate from a single urine sample in patients with E. coli bacteriuria by differential genome quantification (ori:ter) performed by quantitative PCR. We found active bacterial growth in almost all samples. However, this occurs with day-to-day and inter-patient variability. Our observations indicate that chromosome replication provides not only a robust measure of bacterial growth rate, but it can also be used as a means to evaluate antibiotic effect.

Coping with Reactive Oxygen Species to Ensure Genome Stability in Escherichia coli.

The facultative aerobic bacterium Escherichia coli adjusts its cell cycle to environmental conditions. Because of its lifestyle, the bacterium has to balance the use of oxygen with the potential lethal effects of its poisonous derivatives. Oxidative damages perpetrated by molecules such as hydrogen peroxide and superoxide anions directly incapacitate metabolic activities relying on enzymes co-factored with iron and flavins. Consequently, growth is inhibited when the bacterium faces substantial reactive oxygen insults coming from environmental or cellular sources. Although hydrogen peroxide and superoxide anions do not oxidize DNA directly, these molecules feed directly or indirectly the generation of the highly reactive hydroxyl radical that damages the bacterial chromosome. Oxidized bases are normally excised and the single strand gap repaired by the base excision repair pathway (BER). This process is especially problematic in E. coli because replication forks do not sense the presence of damages or a stalled fork ahead of them. As consequence, single-strand breaks are turned into double-strand breaks (DSB) through replication. Since E. coli tolerates the presence of DSBs poorly, BER can become toxic during oxidative stress. Here we review the repair strategies that E. coli adopts to preserve genome integrity during oxidative stress and their relation to cell cycle control of DNA replication.

Chromosome replication as a measure of bacterial growth rate during Escherichia coli infection in the mouse peritonitis model.

The efficacy of most antibiotics is dependent on active bacterial growth, yet little is known about the growth dynamics during infection. Therefore, means to measure in-host bacterial growth rate is of importance. Here, we use chromosome replication as readout for in situ bacterial growth rate during infection; obtained from a single biological specimen. We have applied two independent methods: quantitative PCR (qPCR) and fluorescence microscopy, to quantify the level of chromosome replication present during Escherichia coli propagation in the mouse peritonitis model. We find that the methods complement each other and allow for quantification of growth rate, both on a population average and on a single-cell level. We demonstrate the presence of heterogeneous growth rates within bacterial populations propagating during infection. Also, no growth cessation was observed during the apparent stationary phase in vivo, and, by comparison of growth dynamics at different anatomical sites, we demonstrate that E. coli is unlikely to grow independently intravascularly. These findings provide novel insight into bacterial growth during host infection, and underscore the importance of pinpointing the primary site of infection in septicaemia of unknown origin and ensuring antibiotic availability at this site.

Iron chelation increases the tolerance of Escherichia coli to hyper-replication stress.

In Escherichia coli, an increase in the frequency of chromosome replication is lethal. In order to identify compounds that affect chromosome replication, we screened for molecules capable of restoring the viability of hyper-replicating cells. We made use of two E. coli strains that over-initiate DNA replication by keeping the DnaA initiator protein in its active ATP bound state. While viable under anaerobic growth or when grown on poor media, these strains become inviable when grown in rich media. Extracts from actinomycetes strains were screened, leading to the identification of deferoxamine (DFO) as the active compound in one of them. We show that DFO does not affect chromosomal replication initiation and suggest that it was identified due to its ability to chelate cellular iron. This limits the formation of reactive oxygen species, reduce oxidative DNA damage and promote processivity of DNA replication. We argue that the benzazepine derivate (±)-6-Chloro-PB hydrobromide acts in a similar manner.

Countermeasures to survive excessive chromosome replication in Escherichia coli.

In Escherichia coli, like all organisms, DNA replication is coordinated with cell cycle progression to ensure duplication of the genome prior to cell division. Chromosome replication is initiated from the replication origin, oriC, by the DnaA protein associated with ATP. Initiations take place once per cell cycle and in synchrony at all cellular origins. DnaA also binds ADP with similar affinity as ATP and in wild-type cells the majority of DnaA molecules are ADP bound. In cells where the DnaAATP/DnaAADP ratio increases or in cells where DnaAATP has increased access to oriC, premature initiations take place, often referred to as overinitiation. Overinitiating cells are generally characterized by their slow growth and in the most severe cases lethal accumulation of DNA strand breaks. Here, we review the different strategies adopted by E. coli to survive overinitiation. We propose a unifying model where all mutations that suppress overinitiation keep replication forks separated in time and, thereby, reduce the formation of strand breaks. One group of mutations does so by lowering the activity of oriC and/or DnaA to reduce the frequency of initiations to an acceptable level. In the other group of mutations, replication forks are kept apart by preventing formation of damages that would otherwise cause replication blocks, by allowing bypass of replication blocks and/or by slowing down replication forks. This group of suppressors restores viability despite excessive chromosome replication and provides new insights into mechanisms that safeguard DNA integrity.

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Re-wiring of energy metabolism promotes viability during hyperreplication stress in E. coli.

Chromosome replication in Escherichia coli is initiated by DnaA. DnaA binds ATP which is essential for formation of a DnaA-oriC nucleoprotein complex that promotes strand opening, helicase loading and replisome assembly. Following initiation, DnaAATP is converted to DnaAADP primarily by the Regulatory Inactivation of DnaA process (RIDA). In RIDA deficient cells, DnaAATP accumulates leading to uncontrolled initiation of replication and cell death by accumulation of DNA strand breaks. Mutations that suppress RIDA deficiency either dampen overinitiation or permit growth despite overinitiation. We characterize mutations of the last group that have in common that distinct metabolic routes are rewired resulting in the redirection of electron flow towards the cytochrome bd-1. We propose a model where cytochrome bd-1 lowers the formation of reactive oxygen species and hence oxidative damage to the DNA in general. This increases the processivity of replication forks generated by overinitiation to a level that sustains viability.

Control of bacterial chromosome replication by non-coding regions outside the origin.

Chromosome replication in Eubacteria is initiated by initiator protein(s) binding to specific sites within the replication origin, oriC. Recently, initiator protein binding to chromosomal regions outside the origin has attracted renewed attention; as such binding sites contribute to control the frequency of initiations. These outside-oriC binding sites function in several different ways: by steric hindrances of replication fork assembly, by titration of initiator proteins away from the origin, by performing a chaperone-like activity for inactivation- or activation of initiator proteins or by mediating crosstalk between chromosomes. Here, we discuss initiator binding to outside-oriC sites in a broad range of different taxonomic groups, to highlight the significance of such regions for regulation of bacterial chromosome replication. For Escherichia coli, it was recently shown that the genomic positions of regulatory elements are important for bacterial fitness, which, as we discuss, could be true for several other organisms.

DNA Replication Control Is Linked to Genomic Positioning of Control Regions in Escherichia coli.

Chromosome replication in Escherichia coli is in part controlled by three non-coding genomic sequences, DARS1, DARS2, and datA that modulate the activity of the initiator protein DnaA. The relative distance from oriC to the non-coding regions are conserved among E. coli species, despite large variations in genome size. Here we use a combination of i) site directed translocation of each region to new positions on the bacterial chromosome and ii) random transposon mediated translocation followed by culture evolution, to show genetic evidence for the importance of position. Here we provide evidence that the genomic locations of these regulatory sequences are important for cell cycle control and bacterial fitness. In addition, our work shows that the functionally redundant DARS1 and DARS2 regions play different roles in replication control. DARS1 is mainly involved in maintaining the origin concentration, whether DARS2 is also involved in maintaining single cell synchrony.

Multiple DNA Binding Proteins Contribute to Timing of Chromosome Replication in E. coli.

Chromosome replication in Escherichia coli is initiated from a single origin, oriC. Initiation involves a number of DNA binding proteins, but only DnaA is essential and specific for the initiation process. DnaA is an AAA+ protein that binds both ATP and ADP with similar high affinities. DnaA associated with either ATP or ADP binds to a set of strong DnaA binding sites in oriC, whereas only DnaA(ATP) is capable of binding additional and weaker sites to promote initiation. Additional DNA binding proteins act to ensure that initiation occurs timely by affecting either the cellular mass at which DNA replication is initiated, or the time window in which all origins present in a single cell are initiated, i.e. initiation synchrony, or both. Overall, these DNA binding proteins modulate the initiation frequency from oriC by: (i) binding directly to oriC to affect DnaA binding, (ii) altering the DNA topology in or around oriC, (iii) altering the nucleotide bound status of DnaA by interacting with non-coding chromosomal sequences, distant from oriC, that are important for DnaA activity. Thus, although DnaA is the key protein for initiation of replication, other DNA-binding proteins act not only on oriC for modulation of its activity but also at additional regulatory sites to control the nucleotide bound status of DnaA. Here we review the contribution of key DNA binding proteins to the tight regulation of chromosome replication in E. coli cells.

Secretion of Alpha-Hemolysin by Escherichia coli Disrupts Tight Junctions in Ulcerative Colitis Patients.

OBJECTIVES: The potential of Escherichia coli (E. coli) isolated from inflammatory bowel disease (IBD) patients to damage the integrity of the intestinal epithelium was investigated. METHODS: E. coli strains isolated from patients with ulcerative colitis (UC) and healthy controls were tested for virulence capacity by molecular techniques and cytotoxic assays and transepithelial electric resistance (TER). E. coli isolate p19A was selected, and deletion mutants were created for alpha-hemolysin (α-hemolysin) (hly) clusters and cytotoxic necrotizing factor type 1 (cnf1). Probiotic E. coli Nissle and pathogenic E. coli LF82 were used as controls. RESULTS: E. coli strains from patients with active UC completely disrupted epithelial cell tight junctions shortly after inoculation. These strains belong to phylogenetic group B2 and are all α-hemolysin positive. In contrast, probiotic E. coli Nissle, pathogenic E. coli LF82, four E. coli from patients with inactive UC and three E. coli strains from healthy controls did not disrupt tight junctions. E. coli p19A WT as well as cnf1, and single loci of hly mutants from cluster I and II were all able to damage Caco-2 (Heterogeneous human epithelial colorectal adenocarcinoma) cell tight junctions. However, this phenotype was lost in a mutant with knockout (Δ) of both hly loci (P<0.001). CONCLUSIONS: UC-associated E. coli producing α-hemolysin can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect was abolished in a mutant unable to express α-hemolysin. These results suggest that high Hly expression may be a mechanism by which specific strains of E. coli pathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD.

Control regions for chromosome replication are conserved with respect to sequence and location among Escherichia coli strains.

In Escherichia coli, chromosome replication is initiated from oriC by the DnaA initiator protein associated with ATP. Three non-coding regions contribute to the activity of DnaA. The datA locus is instrumental in conversion of DnaA(ATP) to DnaA(ADP) (datA dependent DnaA(ATP) hydrolysis) whereas DnaA rejuvenation sequences 1 and 2 (DARS1 and DARS2) reactivate DnaA(ADP) to DnaA(ATP). The structural organization of oriC, datA, DARS1, and DARS2 were found conserved among 59 fully sequenced E. coli genomes, with differences primarily in the non-functional spacer regions between key protein binding sites. The relative distances from oriC to datA, DARS1, and DARS2, respectively, was also conserved despite of large variations in genome size, suggesting that the gene dosage of either region is important for bacterial growth. Yet all three regions could be deleted alone or in combination without loss of viability. Competition experiments during balanced growth in rich medium and during mouse colonization indicated roles of datA, DARS1, and DARS2 for bacterial fitness although the relative contribution of each region differed between growth conditions. We suggest that this fitness advantage has contributed to conservation of both sequence and chromosomal location for datA, DARS1, and DARS2.

Oxidative DNA damage is instrumental in hyperreplication stress-induced inviability of Escherichia coli.

In Escherichia coli, an increase in the ATP bound form of the DnaA initiator protein results in hyperinitiation and inviability. Here, we show that such replication stress is tolerated during anaerobic growth. In hyperinitiating cells, a shift from anaerobic to aerobic growth resulted in appearance of fragmented chromosomes and a decrease in terminus concentration, leading to a dramatic increase in ori/ter ratio and cessation of cell growth. Aerobic viability was restored by reducing the level of reactive oxygen species (ROS) or by deleting mutM (Fpg glycosylase). The double-strand breaks observed in hyperinitiating cells therefore results from replication forks encountering single-stranded DNA lesions generated while removing oxidized bases, primarily 8-oxoG, from the DNA. We conclude that there is a delicate balance between chromosome replication and ROS inflicted DNA damage so the number of replication forks can only increase when ROS formation is reduced or when the pertinent repair is compromised.