Bacterial Mutation Research Articles

Tigecycline-Rifampicin Restrains Resistance Development in Carbapenem-Resistant Klebsiella pneumoniae.

The goal of this study was to clarify the synergistic antibacterial activity of the combination of tigecycline (TGC) and rifampicin (RIF). Additionally, the study sought to investigate the impact of this combination on the development of mutational resistance and to assess its efficacy in an in vivo model using Galleria mellonella. Through a checkerboard test, we found that the combination of TGC and RIF showed synergistic antibacterial activity against carbapenem-resistant Klebsiella pneumoniae (CRKP). The fractional inhibition concentration index (FICI) was found to be ≤0.5, confirming the potency of the combination. Additionally, this synergistic effect was further validated in vivo using the G. mellonella infection model. TGC-RIF treatment had a lower mutant prevention concentration (MPC) than that of monotherapy, indicating its potential to reduce the development of mutational resistance. We observed a substantial variation in the MPCs of TGC and RIF when they were measured at different proportions in the combinations. Furthermore, during the resistant mutant selection window (MSW) test, we noticed a correlation between strains with low FICI and low MSW. The expression of efflux-pump-related genes, namely rarA and acrB, is significantly decreased in the combination therapy group. This indicates that altered expression levels of certain efflux pump regulator genes are associated with a combined decrease in bacterial mutation resistance. In conclusion, the combination of TGC and RIF effectively suppresses antibiotic resistance selection in CRKP. This study establishes a paradigm for evaluating drug-resistant mutant suppression in antimicrobial combination therapy.

Flavonoids의 항산화 활성과 미생물 복귀돌연변이에 영향을 미치는 구조적 요인 분석

Flavonoids의 항산화 활성과 미생물 복귀돌연변이에 영향을 미치는 구조적 요인 분석

Genetic characteristics and integration specificity of Salmonella enterica temperate phages.

Temperate phages can engage in the horizontal transfer of functional genes to their bacterial hosts. Thus, their genetic material becomes an intimate part of bacterial genomes and plays essential roles in bacterial mutation and evolution. Specifically, temperate phages can naturally transmit genes by integrating their genomes into the bacterial host genomes via integrases. Our previous study showed that Salmonella enterica contains the largest number of temperate phages among all publicly available bacterial species. S. enterica is an important pathogen that can cause serious systemic infections and even fatalities. Initially, we extracted all S. enterica temperate phages from the extensively developed temperate phage database established in our previous study. Subsequently, we conducted an in-depth analysis of the genetic characteristics and integration specificity exhibited by these S. enterica temperate phages. Here we identified 8,777 S. enterica temperate phages, all of which have integrases in their genomes. We found 491 non-redundant S. enterica temperate phage integrases (integrase entries). S. enterica temperate phage integrases were classified into three types: intA, intS, and phiRv2. Correlation analysis showed that the sequence lengths of S. enterica integrase and core regions of attB and attP were strongly correlated. Further phylogenetic analysis and taxonomic classification indicated that both the S. enterica temperate phage genomes and the integrase gene sequences were of high diversities. Our work provides insight into the essential integration specificity and genetic diversity of S. enterica temperate phages. This study paves the way for a better understanding of the interactions between phages and S. enterica. By analyzing a large number of S. enterica temperate phages and their integrases, we provide valuable insights into the genetic diversity and prevalence of these elements. This knowledge has important implications for developing targeted therapeutic interventions, such as phage therapy, to combat S. enterica infections. By harnessing the lytic capabilities of temperate phages, they can be engineered or utilized in phage cocktails to specifically target and eradicate S. enterica strains, offering an alternative or complementary approach to traditional antibiotic treatments. Our study has implications for public health and holds potential significance in combating clinical infections caused by S. enterica.

Heparin increases the antibiotic efficacy of colistin

The increasing antibiotic resistance in bacteria is an alarming phenomenon all around the world. Certain strains have developed resistance against multiple antimicrobial molecules, in which cases, the final option is to use a last-resort drug. These drugs, however, are last-resort for a reason: they can pose serious risk on vital organ functions in the patient. To mitigate the risk of severe side-effects and to reduce the rate of bacterial mutation, co-administration with other molecules that increase their efficacy seems to be the only suitable option. This leads to a reduced dose while maintaining the same level of antibiotic activity within the body. In this study, the effect of heparin derivatives on the antibiotic activity of colistin and their interactions were studied by ion mobility, mass spectrometry, and bacterium growth assays. The results show that during the association of colistin and heparin, they retain their structure while higher-stoichiometry complexes can form. When long-chain heparin is co-administered, multiple colistin molecules can associate with it, which increases the antibiotic activity by ∼40% relative to the sole administration of colistin.

Investigation of the dual role of acyl phloroglucinols as a new hope for antibacterial and anti-SARS-CoV-2 agents employing integrated in vitro and multi-phase in silico approaches

With the rapid spread of multi-drug-resistant bacteria and more infectious or aggressive variants of SARS-CoV-2, it is critical to develop drugs that can quickly adapt to evolving bacterial and viral mutations. In this study, encouraged by nature, we synthesized a series of phloroglucinol (PG) derivatives, acyl phloroglucinols (ACPLs) 2a-4c by mimicking the structure of the natural antifungal 2,4-diacetylphloroglucinol 3a (2,4-DAPG). According to the quantum chemical calculation, these compounds were expected to be exceptionally favourable for intermolecular interaction with protein receptors. Intriguingly, the in vitro study of Staphylococcus aureus (S. aureus) ATCC 25923 and Escherichia coli (E. coli) ATCC 25922, showed that the four ACPLs 3a–4a had good antibacterial activity and selectivity against gram-positive bacteria, S. aureus. These results were then supported by in silico molecular docking and molecular dynamics simulations (MDs) between these potent compounds and the S. aureus FtsA protein (PDB ID: 3WQU). Furthermore, with the aid of the knowledge base Virus-CKB along with the molecular docking study, it was found that the three ACPLs 4a–4c showed potential inhibitors against SARS-CoV-2 PLpro (PDB ID: 7CMD). Additionally, 100 ns of MDs was carried out in order to assess the stability behaviour of ACPLs at the docked site. Moreover, in silico ADME/T and drug likeness of all studied ACPLs were also predicted. Finally, shape-based screening of FDA-approved drugs was performed using the most prominent synthesized ACPLs as a template, enabling us to include several medications that could be utilized as antibacterial and antiviral drug candidates. Communicated by Ramaswamy H. Sarma

Evaluation of the herbicide glyphosate, (aminomethyl)phosphonic acid, and glyphosate-based formulations for genotoxic activity using in vitro assays.

Glyphosate, the most heavily used herbicide world-wide, is applied to plants in complex formulations that promote absorption. The National Toxicology Program reported in 1992 that glyphosate, administered to rats and mice at doses up to 50,000 ppm in feed for 13 weeks, showed little evidence of toxicity, and no induction of micronuclei was observed in the mice in this study. Subsequently, mechanistic studies of glyphosate and glyphosate-based formulations (GBFs) that have focused on DNA damage and oxidative stress suggest that glyphosate may have genotoxic potential. However, few of these studies directly compared glyphosate to GBFs, or effects among GBFs. To address these data gaps, we tested glyphosate, glyphosate isopropylamine (IPA), and (aminomethyl)phosphonic acid (AMPA, a microbial metabolite of glyphosate), 9 high-use agricultural GBFs, 4 residential-use GBFs, and additional herbicides (metolachlor, mesotrione, and diquat dibromide) present in some of the GBFs in bacterial mutagenicity tests, and in human TK6 cells using a micronucleus assay and a multiplexed DNA damage assay. Our results showed no genotoxicity or notable cytotoxicity for glyphosate or AMPA at concentrations up to 10 mM, while all GBFs and herbicides other than glyphosate were cytotoxic, and some showed genotoxic activity. An in vitro to in vivo extrapolation of results for glyphosate suggests that it is of low toxicological concern for humans. In conclusion, these results demonstrate a lack of genotoxicity for glyphosate, consistent with observations in the NTP in vivo study, and suggest that toxicity associated with GBFs may be related to other components of these formulations.

Novel synthesis of N‐acrylamidociprofloxacin and related polymers: Bioactivity, drug resistance, and drug release

AbstractTwo new ciprofloxacin (CF) monomers, (ACF) and (MCF) were designed and synthesized. Both ACF and MCF were polymerized using a redox‐initiated polymerization process, yielding the polymers PACF and PMCF, respectively. Three co‐polymers were prepared by polymerizing ACF with acrylamide (AcA), acrylonitrile (AcN), or N‐(thiazol‐2‐yl) acrylamide (NTA) monomers in a 1:3 molar ratio. Elemental analysis, X‐ray, IR, 1H and 13C NMR, and mass spectrometry were used to characterize the obtained monomers, polymers, and co‐polymers. The biological activities of the prepared monomers, polymers, and co‐polymers against Gram‐positive and Gram‐negative bacteria were investigated. Newly prepared compounds had a severe‐to‐mild impact on the microorganisms that were tested. Additionally, CF‐resistant bacteria were tested against newly prepared monomers and polyciprofloxacin. These data demonstrated that novel CF derivatives can overcome bacterial mutations that result in resistance to quinolone derivatives. Under acidic and basic conditions at 40°C, the sustained drug release of CF from polymeric chains and co‐polymers was studied. Drug release in the absence and presence of a catalytic quantity of potassium chloride at different concentrations, was studied and showed an increase in extended release. Drug release from the polymer and co‐polymer chains was measured by UV–visible spectroscopy. Morphologies and thermal stability of the prepared polymers and co‐polymers have also been reported.

Mode of constant magnetic power effects on Escherichia coli viability and antibiotics activities

Background: The region that a magnetic force has an effect on is known as a magnetic field. Normally, two poles of this field are concentrated. Most magnetic objects are made up of a variety of tiny fields known as domains. There are many different techniques that have been published in the literature for using magnetic energy as a diagnostic tool and for treating illnesses in both humans and animals. Aims: To investigate the effects of different levels of static magnetic field on the ultra structure of Escherichia coli a bacterium as well as their antibiotics activities changes. Materials and Method: Locally created dipolar static magnetic field with strength 400, 800, 1200, and 1600 Gauss and used. Between July and October 2022, ten patients with urinary tract infections at Hawler Teaching Hospital and Raparren Hospital for Children in Erbil were isolated for E. coli and then identified by Vitek test. Bacterial culture medium in equal amounts of broth was subjected to the magnetic field for 24 hours. Additionally, treated E. coli culture media (Vitek test) was compared with untreated negative control samples in the bacterial growth subculture, which was checked for bacterial population using spectrophotometer and Vitek diagnosis kit depended on response to different types of antibiotics. Results: An recognized bacterial strain known as E. coli was subjected to magnetic field with two poles pressures of (400, 800, 1200, and 1600) Gausses while it was incubated for 24 hours at a temperature of 37oC. Optical density (O.D.) measurements at 620 nm were used. The results showed that the microorganisms' exposure to the magnetic field produced noticeable alterations on response to different types of antibiotics (Ceftazidime, Azetroname, Ceftazidime, Cefepime, Minocyclin, Azetroname, Ticarcillin/ Clavulanic acid, Azetroname, Piperacillin, Ceftazidime, Cefepime, Ciprofloxacin, Tobramycin, Imipenem, Meropenem, Amikacin, Nitrofurantin, Trimethoprim/ Sulfamethoxazole and Gentamycin) and significantly reduced the number of cells in the exposed bacteria as compared to the control. Conclusions: We came to the conclusion that due to bacterial mutation, the magnetic field could alter bacterial response to different types of antibiotics and bacterial population.

Rapid assessment of antibiotic susceptibility using a fully 3D-printed impedance-based biosensor

The sustained misuse and overuse of antibacterial agents is accelerating the emergence of antimicrobial resistance (AMR), which is becoming one of the major threats to public health. Abuse of antibiotics drives spontaneous evolution, bacterial mutation, and exchange of resistant genes through lateral gene transfer. Mitigating the worldwide impact of AMR requires enhanced antibiotic stewardship through faster diagnostic testing. In this work, we aim to tackle this issue via development of a fully 3D-printed electrochemical, gel-modified biosensor for rapid bacterial growth monitoring. By using electrochemical impedance spectroscopy, we have successfully identified growth profiles and confirmed antibiotic susceptibility of two ESKAPE pathogens, Escherichia coli and Pseudomonas aeruginosa, following overnight culture it was possible to determine antibiotic sensitivity in 90 min, altogether faster than the 24–48 h current gold standard of culture-based antimicrobial susceptibility testing with significant scope for optimisation. Results show a clear distinction between growth profiles in the presence and absence of amoxicillin, gentamicin, and fosfomycin, therefore demonstrating a rapid, cost-efficient platform for phenotypic antibiotic susceptibility testing within clinically relevant concentration ranges for conditions such as urinary tract infections and pneumonia.

Overview Methicillin-Resistant Staphylococcus Aureus Combination Treatment Options in Vivo and in Vitro

The spread of multidrug-resistant Staphylococcus aureus continues to threaten global health. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the common causes of bacterial infections in hospitals and communities. Despite vancomycin being an effective treatment for MRSA, from 2006 to 2020, vancomycin-resistant MRSA increased 3.5-fold, from 5% to 7%. Bacterial genome mutations, as well as bacteria’s ability to transfer genetic material with other bacteria, allowing them to obtain resistance genes from different strains, are all factors contributing to the development of vancomycin resistance in MRSA. As a result, combination therapy may be a potential treatment for MRSA infection. We searched in PubMed and Google Scholar, and our search yielded 92 articles, out of which 74 full-text articles were reviewed and 56 were selected for this study. This literature review examines combination therapies for MRSA infections, such as -Lactams with vancomycin, linezolid and imipenem, daptomycin and ceftaroline. The review yielded several studies looking at the synergy between -lactams and vancomycin. Although linezolid and rifampicin demonstrate synergy against MRSA in vivo and in vitro in various invasion diseases, more clinical research is required to prove their efficacy. Furthermore, daptomycin plus ceftaroline shows synergy for refractory staphylococcal bacteremia in vivo and in vitro. Combining ceftaroline and daptomycin has two benefits: they work synergistically together and make the innate host defense peptide cathelicidin leucine-leucine-37 (LL-37) more sensitizing. Ceftaroline plus daptomycin was recently used in MRSA biofilm infections, demonstrating a potentially promising treatment as the first combination used without side effects in humans. Keywords: Bacteremia, Vancomycin, Daptomycin, Ceftaroline, Staphylococcus aureus, MRSA.

Detection of DNA replication errors and 8-oxo-dGTP-mediated mutations in E. coli by Duplex DNA Sequencing

Mutation is a phenomenon inescapable for all life-forms, including bacteria. While bacterial mutation rates are generally low due to the operation of error-avoidance systems, sometimes they are elevated by many orders of magnitude. Such a state, known as a hypermutable state, can result from exposure to stress or to harmful environments. Studies of bacterial mutation frequencies and analysis of the precise types of mutations can provide insights into the mechanisms by which mutations occur and the possible involvement of error-avoidance pathways. Several approaches have been used for this, like reporter assays involving non-essential genes or mutation accumulation over multiple generations. However, these approaches give an indirect estimation, and a more direct approach for determining mutations is desirable. With the recent development of a DNA sequencing technique known as Duplex Sequencing, it is possible to detect rare variants in a population at a frequency of 1 in 107 base pairs or less. Here, we have applied Duplex Sequencing to study spontaneous mutations in E. coli. We also investigated the production of replication errors by using a mismatch-repair defective (mutL) strain as well as oxidative-stress associated mutations using a mutT-defective strain. For DNA from a wild-type strain we obtained mutant frequencies in the range of 10−7 to 10−8 depending on the specific base-pair substitution, but we argue that these mutants merely represent a background of the system, rather than mutations that occurred in vivo. In contrast, bona-fide in vivo mutations were identified for DNA from both the mutL and mutT strains, as indicated by specific increases in base substitutions that are fully consistent with their established in vivo roles. Notably, the data reproduce the specific context effects of in vivo mutations as well as the leading vs. lagging strand bias among DNA replication errors.

Quantification of bacterial adhesion to tissue in high-throughput kinetics.

Bacterial adhesion to tissue is the starting point for many pathogenic processes and beneficial interactions. The dynamics and speed of adhesion (minutes) make high-resolution temporal kinetic data important, but this capability is absent from the current toolset. We present a high-throughput method with a second-to-minute kinetic resolution, testing the adhesion of Pseudomonas aeruginosa PAO1 wild-type, flagella-, pili-, and quorum-sensing mutants to human embryonic kidney (HEK293) cells. Adhesion rates were in good correlation with HEK293 confluence, and the ways in which various bacterial mutations modified adhesion patterns are in agreement with the published literature. This simple assay can facilitate drug screening and treatment development as well as provide a better understanding of the interactions of pathogenic and probiotic bacteria with tissues, allowing the design of interventions and prevention treatments.

A Differential Equation for Mutation Rates in Environmental Coevolution

In their paper Natural selection for least action (Kaila and Annila 2008) they depict evolution as a process conforming to the Principle of Least Action (PLA). From this concept, together with the Coevolution model of Lewontin, an equation of motion for environmental coevolution is derived which shows that it is the time rate (frequency) of evolutionary change of the organism (mutations) that responds to changes in the environment. It is not possible to compare the theory with viral or bacterial mutation rates, as these are not measured on a time base. There is positive evidence from population level avian studies where the coefficient of additive evolvability (CAv) and its square (IA) change with environmental favourability in agreement with this model. Further analysis shows that the time rate of change of the coefficient of additive evolvability (CAv) and its square (IA) are linear with environmental favourability, which could help in defining the Lagrangian of the environmental effects.

Dose-dependent joint resistance action of antibacterial mixtures in their hormetic effects on bacterial resistance based on concentration addition model

The judgment of joint resistance action is significant for evaluating the resistance risk of antibacterial mixture. Using bacterial mutation frequency (MF) and conjugative transfer frequency (CTF) to respectively characterize the bacterial endogenous and exogenous resistance, mutation unit and conjugative transfer unit have been proposed to judge the joint resistance action of antibacterial mixture at a certain dose. However, these methods could not evaluate the antibacterial mixture's joint resistance action at a larger concentration-range. In this study, the concentration addition for bacterial resistance (CA-BR) approach was used to judge the joint resistance actions between kanamycin sulfate (KAN) and some other typical antibacterial agents, including sulfonamides (SAs), sulfonamide potentiators (SAPs), and silver antibacterial compounds (SACs). Through comparing the hormetic dose-response curves of the binary mixtures on the MF (or CTF) in Escherichia coli (E. coli) and the corresponding CA-BR curves calculated from the hormetic dose-responses of the single agents, the joint resistance actions between KAN and other agents were judged to exhibit dose-dependent feature: with the increase of mixture concentration, the joint mutation actions between KAN and SAs (or SAPs) were fixed at synergism, and the joint mutation actions between KAN and SACs varied from antagonism to synergism; the joint conjugative transfer actions between KAN and other agents changed from antagonism to synergism. Mechanistic explanation suggested that the heterogeneous pattern of joint resistance action had a close relationship with the interplays among the agents' modes of action, and meanwhile was significantly influenced by their joint survival pressure on E. coli. This study reveals the dose-dependent feature for the joint resistance action of antibacterial mixture and highlights the importance of exposure concentration, which will benefit clarifying the resistance risk of antibacterial mixture in the environment.

Deciphering the genetic network and programmed regulation of antimicrobial resistance in bacterial pathogens.

Antimicrobial resistance (AMR) in bacteria is an important global health problem affecting humans, animals, and the environment. AMR is considered as one of the major components in the "global one health". Misuse/overuse of antibiotics in any one of the segments can impact the integrity of the others. In the presence of antibiotic selective pressure, bacteria tend to develop several defense mechanisms, which include structural changes of the bacterial outer membrane, enzymatic processes, gene upregulation, mutations, adaptive resistance, and biofilm formation. Several components of mobile genetic elements (MGEs) play an important role in the dissemination of AMR. Each one of these components has a specific function that lasts long, irrespective of any antibiotic pressure. Integrative and conjugative elements (ICEs), insertion sequence elements (ISs), and transposons carry the antimicrobial resistance genes (ARGs) on different genetic backbones. Successful transfer of ARGs depends on the class of plasmids, regulons, ISs proximity, and type of recombination systems. Additionally, phage-bacterial networks play a major role in the transmission of ARGs, especially in bacteria from the environment and foods of animal origin. Several other functional attributes of bacteria also get successfully modified to acquire ARGs. These include efflux pumps, toxin-antitoxin systems, regulatory small RNAs, guanosine pentaphosphate signaling, quorum sensing, two-component system, and clustered regularly interspaced short palindromic repeats (CRISPR) systems. The metabolic and virulence state of bacteria is also associated with a range of genetic and phenotypic resistance mechanisms. In spite of the availability of a considerable information on AMR, the network associations between selection pressures and several of the components mentioned above are poorly understood. Understanding how a pathogen resists and regulates the ARGs in response to antimicrobials can help in controlling the development of resistance. Here, we provide an overview of the importance of genetic network and regulation of AMR in bacterial pathogens.

Risk factors for ventilator-associated pneumonia due to multi-drug resistant organisms after cardiac surgery in adults

BackgroundVentilator-associated pneumonia (VAP) is one of the most common intensive care unit (ICU)-acquired infections, which can cause multiple adverse events. Due to bacterial mutation and overuse of antimicrobial drugs, multidrug-resistant organisms (MDRO) has become one of the major causes of postoperative VAP infections in cardiac patients. Therefore, this study aims to explore the risk factors for VAP with MDRO following cardiac surgery in adults.MethodsThe clinical data of adult VAP patients following cardiac surgery in the hospital from Jan 2017 to May 2021 were analyzed retrospectively, and the patients were divided into the MDRO VAP group and the non-MDRO VAP group. Univariable and multivariable logistic regression analyses were performed on risk factors in patients with MDRO VAP. The species and drug sensitivity of pathogens isolated from the VAP patients were also analyzed.ResultsA total of 61 VAP cases were involved in this study, with 34 cases in the MDRO VAP group (55.7%) and 27 cases in the non-MDRO VAP group (44.3%). Multivariable logistic regression analysis showed that independent risk factors for MDRO VAP included preoperative creatinine clearance rate (CCR) ≥ 86.6ml, intraoperative cardiopulmonary bypass (CPB) time ≥ 151 min, postoperative acute kidney injury (AKI) and nasal feeding. Gram-negative bacilli were the main pathogens in VAP patients (n = 54, 90.0%), with the highest rate of Acinetobacter baumannii (n = 24, 40.0%). Additionally, patients with MDRO VAP had a significantly longer postoperative intensive care unit (ICU) duration and higher hospitalization costs than non-MDRO VAP patients, but there was no notable difference in the 28-day mortality rate between the two groups.ConclusionBased on implementing measures to prevent VAP, clinicians should pay more attention to patients with kidney disease, longer intraoperative CPB time, and postoperative nasal feeding to avoid MDRO infections.

Enterotoxin tilimycin from gut-resident Klebsiella promotes mutational evolution and antibiotic resistance in mice

Klebsiella spp. that secrete the DNA-alkylating enterotoxin tilimycin colonize the human intestinal tract. Numbers of toxigenic bacteria increase during antibiotic use, and the resulting accumulation of tilimycin in the intestinal lumen damages the epithelium via genetic instability and apoptosis. Here we examine the impact of this genotoxin on the gut ecosystem. 16S rRNA sequencing of faecal samples from mice colonized with Klebsiella oxytoca strains and mechanistic analyses show that tilimycin is a pro-mutagenic antibiotic affecting multiple phyla. Transient synthesis of tilimycin in the murine gut antagonized niche competitors, reduced microbial richness and altered taxonomic composition of the microbiota both during and following exposure. Moreover, tilimycin secretion increased rates of mutagenesis in co-resident opportunistic pathogens such as Klebsiella pneumoniae and Escherichia coli, as shown by de novo acquisition of antibiotic resistance. We conclude that tilimycin is a bacterial mutagen, and flares of genotoxic Klebsiella have the potential to drive the emergence of resistance, destabilize the gut microbiota and shape its evolutionary trajectory.

Estimation of Rates of Non-neutral Mutations When Bacteria are Exposed to Subinhibitory Levels of Antibiotics.

The increasingly widespread use of antibiotics has raised an urgent public health question: how does nonlethal exposure of antibiotics increase rates of bacterial mutations that confer antibiotic resistance? Quantitative studies of this sort are rare, and interested investigators tend to neglect scenarios where the mutation inducer and the selective agent are the same. One possible reason for this puzzling neglect is that such an investigation may lead to results that are prone to a Lamarckian interpretation, but a more tangible reason is that successful estimation of mutation rates in such scenarios requires new computational methods. This study presents computational methods tailored for a mutation model in which some wide-type cells may be killed by nonlethal exposure to an antibiotic, but in which mutants proliferate unimpeded. Methods for computing maximum likelihood estimates of mutation rates and their corresponding confidence intervals are devised and evaluated by simulations.

Fitness Trade-Offs in Phage Cocktail-Resistant Salmonella enterica Serovar Enteritidis Results in Increased Antibiotic Susceptibility and Reduced Virulence.

ABSTRACTThe rapid emergence of phage-resistant bacterial mutants is a major challenge for phage therapy. Phage cocktails have been considered one approach to mitigate this issue. However, the synergistic effect of randomly selected phages in the cocktails is ambiguous. Here, we rationally designed a phage cocktail consisting of four phages that utilize the lipopolysaccharide (LPS) O antigen, the LPS outer core, the LPS inner core, and the outer membrane proteins BtuB and TolC on the Salmonella enterica serovar Enteritidis cell surface as receptors. We demonstrated that the four-phage cocktail could significantly delay the emergence of phage-resistant bacterial mutants compared to the single phage. To investigate the fitness costs associated with phage resistance, we characterized a total of 80 bacterial mutants resistant to a single phage or the four-phage cocktail. We observed that mutants resistant to the four-phage cocktail were more sensitive to several antibiotics than the single-phage-resistant mutants. In addition, all mutants resistant to the four-phage cocktail had significantly reduced virulence compared to wild-type strains. Our mouse model of Salmonella Enteritidis infection also indicated that the four-phage cocktail exhibited an enhanced therapeutic effect. Together, our work demonstrates an efficient strategy to design phage cocktails by combining phages with different bacterial receptors, which can steer the evolution of phage-resistant strains toward clinically exploitable phenotypes.IMPORTANCE The selection pressure of phage promotes bacterial mutation, which results in a fitness cost. Such fitness trade-offs are related to the host receptor of the phage; therefore, we can utilize knowledge of bacterial receptors used by phages as a criterion for designing phage cocktails. Here, we evaluated the efficacy of a phage cocktail made up of phages that target four different receptors on Salmonella Enteritidis through in vivo and in vitro experiments. Importantly, we found that pressure from phage cocktails with different receptors can drive phage-resistant bacterial mutants to evolve in a direction that entails more severe fitness costs, resulting in reduced virulence and increased susceptibility to antibiotics. These findings suggest that phage cocktail therapy using combinations of phages targeting different important receptors (e.g., LPS or the efflux pump AcrAB-TolC) on the host surface can steer the host bacteria toward more detrimental surface mutations than single-phage therapy, resulting in more favorable therapeutic outcomes.

Preclinical toxicological assessment of levothyroxine and liothyronine Maillard impurities.

Following the introduction of new stability-indicating related substances methods, an unknown impurity was observed in levothyroxine (LeMI) and liothyronine (LiMI) tablets (ADVANZ PHARMA) in concentrations ≥1.0%, from 6months of storage onwards. The impurity was identified as a Maillard condensation product between lactose and LeMI/LiMI in the LeMI and LiMI tablets, respectively. To establish the toxicity profile of LeMI and LiMI in humans and to define appropriate shelf-life specification limits, a comprehensive nonclinical toxicological assessment was performed, including in silico (Leadscope and Derek Nexus analyses), in vitro (Ames test), and in vivo tests (7-day dose range finding and 90-day dose repeat studies in rats). In silico analyses indicated that potential LeMI and LiMI structures should not be considered bacterial mutagens or in vitro/in vivo clastogens, and that at the low oral exposure levels expected, the impurities are unlikely to cause harm. In vitro testing showed that neither LeMI nor LiMI were cytotoxic or mutagenic at up to 5000μg/plate, both in the presence and absence of metabolic activation. The 2 in vivo studies further confirmed that no systemic toxicity or other notable negative effects were evident at up to 200μg/kg/day for LeMI and 45μg/kg/day for LiMI, the highest doses tested. These doses represent 120-122 times the maximum daily exposures of LeMI and LiMI, based on body surface area (μg/m2). Based on these results, a proposal has been formulated to increase the limits of Maillard condensation products to ≤8.0% for LeMI and≤6.0% for LiMI at shelf life.