Antibiotics

ObjectivesTargeted therapies seek to selectively eliminate a pathogen without disrupting the resident microbial community. This is even more important when a pathogen like H. pylori resides in stomach, a sensitive microbial ecosystem. Using a probiotic like Lactococcus lactis and bioengineering it to release a guided Antimicrobial Peptide (AMP) targeted towards the pathogen offers a pathway to specifically knock-out the deleterious species and not disturbing the stomach microbiome. ResultsThree AMPs, Alyteserin, CRAMP and Laterosporulin, were genetically fused to a guiding peptide MM1, which selectively binds to Vacuolating Toxin A (VacA) of H. pylori and cloned into an excretory vector pTKR inside L. lactis. When cultured together in vitro, the L. lactis bioengineered with guided AMPs selectively killed H. pylori when compared to E. coli or Lactobacillus plantarum, as determined by qPCR. Chemically synthesized Alyteserin and MM1-Alyteserin showed similar preferential inhibition of H. pylori when compared against E. coli, with the MIC of MM1-Alyteserin becoming significantly higher for E. coli than Alytserin whereas no such effet was observed against H. pylori. ConclusionsProbiotics bioengineered to excrete guided AMPs can be a novel and useful approach for combating pathogens without endangering the natural microbial flora. Given the wealth of AMPs and guiding ligands, both natural and synthetic, this approach can be adapted to develop a diverse array of chimeric guided AMPs and can be cloned into probiotics to create a safe and effective alternative to conventional chemical antibiotics.

ObjectivesTargeted therapies seek to selectively eliminate a pathogen without disrupting the resident microbial community. This is even more important when a pathogen like H. pylori resides in stomach, a sensitive microbial ecosystem. Using a probiotic like Lactococcus lactis and bioengineering it to release a guided Antimicrobial Peptide (AMP) targeted towards the pathogen offers a pathway to specifically knock-out the deleterious species and not disturbing the stomach microbiome. ResultsThree AMPs, Alyteserin, CRAMP and Laterosporulin, were genetically fused to a guiding peptide MM1, which selectively binds to Vacuolating Toxin A (VacA) of H. pylori and cloned into an excretory vector pTKR inside L. lactis. When cultured together in vitro, the L. lactis bioengineered with guided AMPs selectively killed H. pylori when compared to E. coli or Lactobacillus plantarum, as determined by qPCR. Chemically synthesized Alyteserin and MM1-Alyteserin showed similar preferential inhibition of H. pylori when compared against E. coli, with the MIC of MM1-Alyteserin becoming significantly higher for E. coli than Alytserin whereas no such effet was observed against H. pylori. ConclusionsProbiotics bioengineered to excrete guided AMPs can be a novel and useful approach for combating pathogens without endangering the natural microbial flora. Given the wealth of AMPs and guiding ligands, both natural and synthetic, this approach can be adapted to develop a diverse array of chimeric guided AMPs and can be cloned into probiotics to create a safe and effective alternative to conventional chemical antibiotics.

IntroductionThe emergence and spread of antibiotic resistance is on the rise around the world, posing a serious threat to public health in the twenty-first century. Several research conducted in various nations have found that the general public plays a pivotal role in the increase and spread of antibiotic resistance. The present study was designed to determine the patient knowledge and perception about antibiotics in community pharmacy. Methods200 participants were recruited by convenience sampling from patients visiting the pharmacy with a prescription for antibiotics and those fulfilling the eligibility criteria for this research. A structured questionnaire was used to access the patients knowledge and perception regarding antibiotics. Data collected were analyzed using Microsoft excel and Epi Info Software version 7 which was used to determine predictors of low antibiotic knowledge. ResultsOverall, 200 questionnaires were analyzed. 70.5% of the respondents had an intermediate level of knowledge. Misconceptions that antibiotics would work on viral infections were reported. 82% of the respondents could correctly identify that misuse of antibiotics can cause antibiotic resistance. The age, educational level, and whether or not the participants were studying or working in medical field were found to be important predictors of antibiotic knowledge. ConclusionThe findings of this study demonstrate that the public surveyed has misunderstandings and a lack of knowledge in some crucial aspects of prudent antibiotic use. Also, negative attitudes regarding rational use of antibiotics were evident. Educational interventions are required to promote rationale use of antibiotics among the general public.

The increasing number of antibiotic resistant bacterial infections is a global threat to human health. Antibiotic resistant bacterial strains generally evolve from susceptible strains by either horizontal gene transfer or chromosomal mutations. After evolving within a host, such resistant strains can be transmitted to other hosts and increase in frequency in the population at large. Population genetic theory postulates that the increase in frequency of an adaptive trait can lead to signatures of selective sweeps. One would thus expect to observe reduced genetic diversity amongst that part of the population that carries the adaptive trait. Specifically, if the evolution of new resistant strains is rare, it is expected that resistant strains represent only a subset of the diversity of susceptible strains. It is currently unknown if diversity of resistant strains is indeed lower than diversity of susceptible strains when considering antibiotic resistance. Here we show that in several bacterial species in several different datasets, sequence-type diversity amongst antibiotic-resistant bacterial strains is indeed lower than amongst antibiotic-susceptible strains in most cases. We re-analysed eight existing clinical datasets with Escherichia coli, Staphylococcus aureus and Enterococcus faecium samples. These datasets consisted of 53 - 1094 patient samples, with multi-locus sequence types and antibiotic resistance phenotypes for 3 - 19 different antibiotics. Out of 59 comparisons, we found that resistant strains were significantly less diverse than susceptible strains in 51 cases (86%). In addition, we show that sequence-type diversity of antibiotic-resistant strains is lower if resistance is rare, compared to when resistance is common, which is consistent with rare resistance being due to fewer evolutionary origins. Our results show that for several different bacterial species, we observe reduced diversity of resistant strains, which is consistent with the evolution of resistance driven by selective sweeps stemming from a limited number of evolutionary origins. In future studies, more detailed analysis of such sweep signatures is warranted.

The antagonistic relationship between bacteria and bacteriophages (phages) drives genetic changes that result in phage resistance. Phage resistance mutations arise in natural microbial communities and during the treatment of diseases with phages (phage therapy), making it important to understand the dynamics of resistance acquisition. It is well-established that when bacteria are challenged with phages in dense liquid cultures, bacterial populations quickly become dominated by phage-resistant variants. However, these conditions--well-mixed liquid cultures with high phage concentrations--are not necessarily common in microbial ecosystems. We developed a simplified mathematical model of phage resistance evolution to explore how phage and host concentration impact the dynamics of resistance evolution. The model was parameterized with microbial growth data from two pathogens and their phages: Pseudomonas aeruginosa and Paenibacillus larvae. Our analyses revealed two fundamental discoveries about resistance evolution. First, phage resistance evolution is predictably governed by a core set of parameters that exhibited high resolution across all bacterial strains: intrinsic growth rate of susceptible bacteria, resistance acquisition rate, fitness cost of resistance, and phage adsorption rate. Second, competitive interactions and fitness costs are the primary drivers of resistance patterns rather than intrinsic mutation rates. We observed three distinct growth patterns--delayed growth, two phase growth, and complete suppression--corresponding to specific parameter regimes as initial phage concentration increased. Two phase growth patterns emerged when competitive dynamics remained balanced, enabling coexistence of susceptible and resistant populations. Complete suppression patterns occurred when low proliferation thresholds combined with extreme competitive asymmetries created unsustainable conditions for resistant bacteria. These findings demonstrate that phage resistance evolution is fundamentally an ecological process where competitive context determines outcomes independently of mutation capacity, with important implications for phage therapy design.

BackgroundStaphylococcus aureus is a gram-positive bacterium that can cause various diseases and infections. Penicillin and methicillin are examples of {beta}-lactam antibiotics, the first line of defense against Staphylococcus aureus infections. Methicillin-Resistant Staphylococcus aureus (MRSA) is still one of the leading causes of hospital-acquired infections associated with morbidity, mortality, and cost. MRSA can be hospital-acquired (HA-MRSA) or community-associated (CA-MRSA) infections. The main objective of this study is to screen MRSA among HA-MRSA to determine the prevalence of antibiotic susceptibility patterns of MRSA among patients. Furthermore, we identify the mecA gene, which produces a penicillin-binding protein (PBP2a) with a low affinity for {beta}-lactam antibiotics. MethodsThis study was done on the patients of Kathmandu Model Hospital, Nepal, and the samples were processed at the Microbiology laboratory of Kathmandu Model Hospital. Data analyses were done from Microsoft Excel and GraphPad Prism. DNA extraction was done from the classical CTAB method with minor modifications, and mecA gene-specific primers were used to detect the gene in the samples. ResultsOut of 4383 samples, 848 (21.00%) samples have growth, and 190(22.4%) were Staphylococcus aureus. Among Staphylococcus aureus 52 (27.36%) were Methicillin resistant Staphylococcus aures. Antibiotic susceptibility tests were done to characterize MRSA isolates. Most of the isolates were resistant to Amikacin (69.25%), followed by Ampicillin (53.8%), Chloramphenicol (78.84%), Cotrimoxazole (53.8%), Gentamycin (67.3%), Ofloxacin (15.39%), Erythromycin (71.15%) Vancomycin and Teicoplanin (3.84%). In our study, 50 (96.15%) out of 52 MRSA showed the mecA gene, while 3.85% showed the absence of the mecA gene. ConclusionsThe frequency of MRSA infections in HA-MRSA was comparatively high, with a greater abundance of the mecA gene that confers the resistance. Regular surveillance of HA-MRSA and genetic profiling of the mecA gene are essential for reducing MRSA infection.

Persister cells are bacterial cells that form small subpopulations and are refractory to antimicrobials. Importantly, those cells can cause the relapse of an infection and also provide a starting point for antimicrobial resistance evolution. By now, several mechanisms of persister formation have been studied. For example, it has been shown that a short exposure to sublethal concentrations of antimicrobials can induce persister formation. Given this, we hypothesised that the pharmacokinetics, the temporal changes of drug concentrations under treatment, could impact persister formation. We predicted that the longer bacteria spend under sublethal drug concentrations, the more persisters would form. Using a set-up with small chemostats and the antimicrobial peptide Pexiganan, we indeed found that faster pharmacokinetics resulted in lower persister numbers compared to slower pharmacokinetics. This finding provides a proof of principle that pharmacokinetics, which can be influenced by treatment procedures, has consequences for persister formation. Our results suggest that faster pharmacokinetics could be useful to minimize persister numbers and hence to lower the risks of relapse or resistance evolution.

Increasing antibiotic resistance poses a serious threat, especially in patients admitted to ICUs. The use of phages in combination with antibiotics as compassionate therapy has become a choice of treatment for pan-drug-resistant bacteria. Here, we studied the cumulative effect of phages with four antibiotics, fosfomycin, ciprofloxacin, vancomycin and oxacillin using three different treatment orders against S. aureus. The antibiotic disc synergy method showed that the plaque size of the phage increased in the subinhibitory antibiotic zone. The sub-inhibitory antibiotic amended in the agar media showed that the plaque size increased between 0.25 g/mL and 1 g/mL of antibiotics. It increases from 0.5 {+/-} 0.1 mm (phage-alone control) to 4 {+/-} 0.2 mm, 1.6 {+/-} 0.1 mm, and 1.6 {+/-} 0.4 mm with fosfomycin, ciprofloxacin, and oxacillin, respectively. Checkerboard analysis showed that phages and antibiotics were synergistic with the FIC index of less than 0.5. So, phage-antibiotic combination treatment appeared to be effective. However, the highest efficiency was observed when the antibiotics were administered after phage treatment. A maximum of 39.4-, 39.4-, and 37.0-fold reduction relative to untreated bacterial culture was observed with fosfomycin, oxacillin, and ciprofloxacin. Vancomycin antibiotic had a least 14.7-fold reduction. Finally, our findings emphasize the potential benefits of phage-antibiotic combination therapy compared with phage-alone therapy to treat S. aureus infections.

The mutation is a fundamental source of biological evolution that create genetic variation in populations. Mutations can create new advantageous traits, but also potentially interfere with pre-existing organismal functions. Therefore, organisms may have evolved their mutation rates to appropriate levels to maintain or improve their fitness. In this study, we aimed to experimentally quantify the relationship between mutation rate and the speed of antibiotic resistance evolution. We conducted experimental evolution using twelve Escherichia coli mutator strains with increased mutation rates and five antibiotics. Our results showed that the highest mutation rate did not necessarily lead to the highest speed of adaptation, indicating a non-monotonic relationship between the speed of drug resistance evolution and mutation rate as expected. Moreover, this relationship was observed to differ among drugs, with significant differences in peak size observed between bacteriostatic and bactericidal antibiotics. We also successfully reproduced the mutation-rate dependence of the speed of adaptation using numerical simulation of a population dynamics model. These findings offer significant insights into the mutation rates evolution concomitant with the development of antibiotic resistance.

Enterococci resistance is increasing sharply, which poses a serious threat to public health. Rhamnolipids are a kind of amphiphilic compound used for its bioactivities, while the combination of nontraditional drugs to restore linezolid activity is an attractive strategy to treat infections caused by these pathogens. This study aimed to investigate the activity of linezolid in combination with the rhamnolipids against Enterococcus faecium. Here, we determined that the rhamnolipids could enhance the efficacy of linezolid against enterococci infections by a checkerboard MIC assay, a time-kill assay, a combined disk test, anti-biofilm assay, molecular simulation dynamics, and mouse infection models. We identified that the combination of rhamnolipids and linezolid restored the linezolid sensitivity. Anti-biofilm experiments show that our new scheme can effectively inhibit biofilm generation. The mouse infection model demonstrated that the combination therapy significantly reduced the bacterial load in the feces, colons and kidneys following subcutaneous administration. This study showed that rhamnolipids could play a synergistic role with linezolid against Enterococcus. Our combined agents could be appealing candidates for developing new combinatorial agents to restore antibiotic efficacy in the treatment of linezolid-resistant Enterococcus infections. HighlightThe identification of a potential inhibitor of LRE, RLS could restore the antibacterial activity of LNZ. A rational mechanism of synergism between LNZ and RLS. ;;L;;RLS in combination with LNZ promote the egress of pathogenic E. faecium from the intestinal tract of mice to reduce its colonization, which has obvious synergistic effects.

BackgroundVirtual colony count is a kinetic, 96-well turbidimetric assay that has been used since 2003 to determine the antimicrobial activity of antimicrobial peptides including the defensin HNP1. Virtual colony count results differed from traditional colony counting results in studies of the antimicrobial activity of the human cathelicidin LL-37 and related peptides. The difference could possibly have been caused by an inoculum effect. MethodsThe virtual colony count assay was conducted using inocula that varied from 1250 to 1x108 virtual colony forming units (CFUv) per milliliter. ResultsThe virtual colony count assay demonstrated a pronounced inoculum effect of HNP1 against Staphylococcus aureus ATCC 29213, accompanied by biofilm formation observed in the wells of the 96 well plates at all inocula. The S. aureus inoculum effect was not as drastic as previously reported for Escherichia coli. ConclusionsThe inoculum effect is further evidence that biofilm formation is a resistance mechanism used by a variety of bacteria against antimicrobial peptides such as HNP1.

Antibiotic resistant microbial pathogens are becoming a major threat to human health. Therefore, there is an urgent need to develop new alternatives to conventional antibiotics. One such promising alternative is antimicrobial peptides (AMPs), which are produced by virtually all organisms and typically inhibit bacteria via membrane disruption. However, previous studies demonstrated that bacteria can rapidly develop AMP resistance. Here, we study whether combination therapy, known to be able to inhibit the evolution of resistance to conventional antibiotics, can also hinder the evolution of AMP resistance. To do so, we evolved the opportunistic pathogen S. aureus in the presence of individual AMP, AMP pairs, and a combinatorial antimicrobial peptide library. Treatment with some AMP pair indeed hindered the evolution of resistance compared with individual AMPs. In particular, resistance to pairs was delayed when resistance to the individual AMPs came at a cost of impaired bacterial growth, and did not confer cross-resistance to other tested AMPs. The lowest level of resistance evolved during treatment with the combinatorial antimicrobial peptide library termed random antimicrobial peptide mixture, which contains more than a million different peptides. A better understanding of how AMP combinations affect the evolution of resistance is a crucial step in order to design resistant proof AMPs cocktails that will offer a sustainable treatment option for antibiotic resistant pathogens.

The scarcity of antibiotics and the need for swift decision-making present significant challenges for healthcare practitioners. When confronted with such circumstances, practitioners must prioritize their approach based on several key factors. By leveraging the recent biological discovery of collateral sensitivity, we have devised an open-source computational platform. This platform utilizes the drug resistance profile of an evolved strain and initial population conditions to potentially predict the failure of cycling therapy in eradicating or restraining a multi-drug resistant bacterial population. We demonstrate how this framework can anticipate potential failures for a range of antibiotics in chronic pseudomonas aeruginosa infections. This innovative methodology lays the foundation for evolutionary therapies that can facilitate the selection of appropriate treatments, thereby reducing antibiotic resistance.

The multidrug resistance (MDR) problem in Streptococcus suis(S. suis) is becoming increasingly severe, necessitating the development of novel antibacterial agents and strategies. In this study, seven Elaiophylin derivatives were isolated from Streptomyces sp. WS-30248, and the antibacterial activity and mechanism of action of the principal compound, Elaiophylin, against S. suis were systematically evaluated for the first time. Through comprehensive approaches including in vitro efficacy assays, biofilm inhibition and eradication tests, bacterial membrane integrity analysis, reactive oxygen species (ROS) level detection, and a mouse infection model, Elaiophylin was found to exhibit significant antibacterial activity against multiple clinically MDR S. suis strains. Its minimum inhibitory concentration (MIC) was as low as 0.5 g/mL, and complete bactericidal activity was achieved within 24 hours at 4xMIC. The compound effectively inhibited and eradicated bacterial biofilms, directly killing embedded cells within the biofilm matrix. Mechanistic studies revealed that Elaiophylin functions through multiple synergistic pathways, including disruption of bacterial membrane integrity and induction of massive ROS accumulation, thereby interfering with the proton motive force (PMF), depleting intracellular ATP, and blocking energy metabolism. In the mouse infection model, the Elaiophylin-treated group showed a significantly increased survival rate of 60% and effectively reduced bacterial loads in tissues. This study demonstrates that Elaiophylin is a highly promising natural candidate drug with multi-target synergistic effects, offering a new strategy to combat MDR S. suis and biofilm-associated infections.

The global surge in multidrug-resistant (MDR) bacterial pathogens has created an urgent imperative for innovative antimicrobial strategies. Enterococcus faecalis, Staphylococcus aureus, and Acinetobacter baumannii demonstrate remarkable antibiotic resistance and dominate hospital-acquired infections. These bacteria often form biofilms, a complex community structure that shields them from immune system phagocytosis, resists antibiotic penetration, and enhances their survival in harsh environments. In clinical cases, these bacteria often form mixed biofilms and lead to treatment failures. Phages and their derivatives have emerged as promising candidates in the fight against drug-resistant bacteria. Lys22, an endolysin derived from an enterococcus phage, has been cloned and demonstrated to possess a broad host range, effectively targeting E. faecalis, various Staphylococcus species, and A. baumannii. When applied to the biofilms formed by these bacteria, Lys22 was found to significantly inhibit both simple and complex biofilms in vitro. Virulent genes, including agrA, sarA, and icaA in S. aureus; asa1, cylA, and hyl in E. faecalis; and OmpA and lpsB in A. baumannii were also downregulated by Lys22. Notably, Lys22 also exhibited a robust protective effect against dual or triple infections involving E. faecalis, S. aureus, and A. baumannii in a zebrafish eggs model, highlighting its potential as a therapeutic agent in combatting multi-bacterial infections.

The urgent necessity for new antibiotics becomes glaringly evident with the relentless rise of multidrug-resistant (MDR) Acinetobacter baumannii in clinical environments, where its infections lead to alarmingly high mortality rates. Antimicrobial peptides (AMPs) represent a promising novel option to combat nosocomial infections caused by MDR A. baumannii. In this study, six novel synthetic peptides were designed through generative artificial intelligence (AI) and synthesized for further experiments. Peptides AIG-R1, AIG-R4, and AIG-R5 showed potent broad-spectrum antibacterial activity against Gram positive and Gram negative pathogens. One of the peptides, AIG-R5, was effective even against colistin and carbapenem-resistant strains of A. baumannii, prevented biofilm formation, and eradicated established biofilms by 60%. Notably, AIG-R5 enhanced the activity of different antibiotics and was found to exhibit synergistic activity with antibiotics from the Aminoglycoside class. The combination of AIG-R5 and Tobramycin at 1/8xMIC and 1/4xMIC effectively reduced pre-formed biofilms of carbapenem resistant A. baumannii more than either component alone, as documented by confocal laser scanning microscopy (CLSM). Significant dose reduction and negligible cytotoxicity exhibited by AIG-R5 with aminoglycosides further encourages evaluation of the combinations therapeutic potential in vivo against MDR A. baumannii infections.

BackgroundAntimicrobial resistance (AMR) is a global crisis, causing 2.8 million infections and 35,000 deaths annually. Staphylococcus aureus is mainly responsible for causing these challenging infections through biofilm formation and the action of efflux pumps. A limited number of studies on Hop (Humulus lupulus) have shown its potential to inhibit quorum sensing in pathogenic bacteria. ObjectiveTherefore, a novel treatment approach was used in this study, which investigated Hops {beta}-acids, particularly the combination of colupulone and n+adlupulone, as well as in combination with fluoroquinolone antibiotics ciprofloxacin and ofloxacin. As ciprofloxacin remains a highly effective antibiotic against Staphylococcus aureus but resistance can develop, and ofloxacin exhibits naturally higher resistance in S. aureus, this study hypothesized that combining Hop (containing colupulone & n+adlupulone) with the two antibiotics separately would result in a greater reduction in biofilm growth of S. aureus compared to their individual potency levels. MethodsAntimicrobial activity was assessed using disk diffusion assays and minimum inhibitory concentration for biofilms at multiple concentrations through 2-fold serial dilutions. ResultsOur data demonstrate that Hop-derived {beta}-acids possess direct antimicrobial activity and when combined with the fluoroquinolone antibiotics, exhibit additive or synergistic effects by acting on different targets in Staphylococcus aureus. ConclusionsThis study provides insight into how natural products can potentially mitigate the development of resistance to antibiotics like ciprofloxacin in the highly pathogenic bacterium S. aureus. It also highlights how adding natural compounds could improve drug effectiveness. Therefore, this demonstrates the potential of natural compounds and antibiotics like ofloxacin, which are known to be ineffective against S. aureus. It offers a promising natural-conventional hybrid approach to addressing antimicrobial resistance.

Antimicrobial resistance (AMR) poses a significant threat to global public health. Notably, resistance to carbapenem and extended-spectrum {beta}-lactam antibiotics in Gram-negative bacteria is a major impediment for the treatment of infections. Genes responsible for resistance to these antibiotics are frequently carried on plasmids, which can transfer between bacteria. Therefore, exploring strategies to prevent this transfer and/or the prevalence of AMR plasmids is timely and pertinent. Here, we show that certain natural product extracts and associated pure compounds can reduce the transmission of AMR plasmids into new bacterial hosts. Using our established high-throughput fluorescence-based screen we found that the natural products were more active in reducing transmission of the IncK plasmid pCT in Escherichia coli ST131, compared to Klebsiella pneumoniae Ecl8 carrying the IncFII plasmid pKpQIL. Furthermore, we found that the natural product rottlerin was more active in K. pneumoniae than in E. coli. Importantly, rottlerin was also associated with a reduced number of transconjugant bacteria in a clinical K. pneumoniae isolate harbouring a blaNDM-1 plasmid. Together, these results demonstrate the potential of natural products as promising anti-plasmid agents.

Antibiotic resistance in bacteria is suggested to be the greatest risk to human health, but new agents are not being brought to market as the rapid evolution of resistance to them means that drug development costs cannot be recouped. Fatty acids have been proposed as a new generation of antibiotics, but toxicity and poor absorption has meant that their use has been impractical in the past. Chitosan has been used to encapsulate other agents as nanoparticles, but has not been used with fatty acids. Here we show that chitosan can be modified to direct fatty acids towards Gram-positive or negative bacteria so that they exert antimicrobial effects. We show that fatty acids work as effective antibiotics in vitro and in vivo, with activity against extremely drug resistant pathogens. Bacteria exposed to them do not develop resistance to these agents, and they are not toxic to mammalian cells. Activity was seen against salmonellosis and C. difficile infection in animal models. Our results demonstrate that fatty acids formulated as chitosan nanoparticles are effective antibiotics, and can be used for a long period of time without resistance developing. This suggests that the usage of fatty acids coated in this manner could be sold in sufficient quantities to recoup its development costs, overcoming this barrier. These agents would form a new class of antibiotics, with the novel property of lack of bacterial resistance.

Antimicrobials can select for antimicrobial resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically stable and very persistent, recirculation of sub-inhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens. The persistence of three antimicrobials over time and their potential effects on antimicrobial resistance were determined in four groups of broilers. Groups were left untreated (control) or were treated with amoxicillin (non-persistent), doxycycline or enrofloxacin (persistent). Antimicrobials were extracted from the faecal samples and concentrations were measured by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism. Up to 37 days after treatment, persistent antimicrobials (doxycycline and enrofloxacin) had concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the non-persistent (amoxicillin) treatment. The amoxicillin treatment showed a significant difference (p [≤] 0.01 and p [≤] 0.0001) in the genotypic resistance only directly after treatment. On the other hand, the doxycycline treatment showed approximately 52% increase in phenotypic and a significant difference (p [≤] 0.05 and p [≤] 0.0001) in genotypic resistance throughout the trial. Furthermore, the enrofloxacin treatment resulted in a complete enrofloxacin-resistant E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations. Based on our findings, we suggest that persistency of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock.