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.

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.

BackgroundAntimicrobial resistance (AMR) is a global public health threat driven significantly by antimicrobial misuse in agriculture, particularly in poultry farming. This study assessed the awareness, knowledge, practices, and associated factors related to antimicrobial resistance among poultry farmers in Osun State, Nigeria. MethodsA cross-sectional study was conducted among 289 poultry farmers selected through stratified random sampling across Osun State. The study included actively practicing poultry farmers aged 18 years and above who used antimicrobials in their operations. Farmers not using antimicrobials were excluded. Data were collected using a pre-tested, structured, interviewer-administered questionnaire and analyzed with SPSS version 27. Descriptive statistics, chi-square tests, and inferential analyses were used to examine relationships between variables. ResultsThe majority of respondents (89.6%) had heard of AMR, the majority 239 (92.3%) of the respondents heard it from veterinary doctors. The majority (77.2%) also demonstrated good knowledge. Most farmers (89.6%) used antibiotics, with 52.9% using them occasionally. Personal experience (57.8%) was the primary basis for antibiotic selection. About 71.6% implemented biosecurity measures, and 57.8% had received training on AMR. Significant associations were found between knowledge and practice (p<0.001) and between attitude and practice (p<0.001). ConclusionDespite high awareness, antibiotic misuse persists, driven by factors such as reliance on personal experience and limited veterinary consultation. There is a need for enhanced farmer education, stricter regulatory enforcement, and the implementation of targeted antimicrobial stewardship programs to mitigate AMR risks in poultry farming.

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.

BackgroundThe emergence of antimicrobial resistance is growing human and animal health concern around the world. When a number of studies have emphasized the Knowledge, Attitude and Practice (KAP) regarding antibiotic use and resistance in humans, little attention has been paid to the veterinary sector. The aim of this study was to understand the KAP towards antibiotic use and resistance among the veterinarians in Bangladesh. MethodsA cross-sectional online based questionnaire survey was conducted from August to September 2020 among the registered veterinary practitioners. A self-administered Google form questionnaire consists of 45 questions on knowledge, attitude and practice regarding antibiotic use and resistance were used. ResultsA total of 208 registered veterinarians participated in this study. 85.1% of the participants were male and 54.8% of the participants had a Masters degree. Around 52% of the veterinarians were poultry practitioners. All respondents were familiar with antimicrobials. The participants (91.4%) knew that antibiotics can not cure viral infections while 97.6% believed that frequent antibiotic prescription rendered them less effective. Participants claimed that only they are eligible to prescribe drugs for the treatment of animals and around 80% disagreed with adding antibiotics with feed/water as a growth promoter in livestock. Of the total participants, 87% believed that a local antimicrobial guideline would be more effective than an international one. However, gaps in practices were highlighted in our study, suggesting training deficiencies. ConclusionThe present study for the first time conducted in Bangladesh dictates the future interventions like academic courses, workshops, and seminars on antibiotic usage and resistance are needed to ameliorate the knowledge, behavior and practice of veterinarians with regards to the rational use of antibiotics.

BackgroundRecent studies showed promising results on application of iodine-containing nanomicelles, FS-1, against antibiotic resistant pathogens. The effect was studied on Escherichia coli ATCC BAA-196. Materials & methodsRNA sequencing for transcriptomics and the complete genome sequencing by SMRT PacBio RS II technology followed by genome assembly and methylomics study were performed. Results & conclusionsFS-1 treated E. coli showed an increased susceptibility to antibiotics ampicillin and gentamicin. The analysis of differential gene regulation showed that possible targets of iodine-containing particles are cell membrane fatty acids and proteins, particularly cytochromes, that leads to oxidative, osmotic and acidic stresses. Cultivation with FS-1 caused gene expression alterations towards anaerobic respiration, increased anabolism and inhibition of many nutrient uptake systems. Identification of methylated nucleotides showed an altered pattern in the FS-1 treated culture. Possible role of transcriptional and epigenetic modifications in the observed increase in susceptibility to gentamicin and ampicillin were discussed. Lay abstractNew approaches of combatting drug resistance infections are in demand as the development of new antibiotics is in a deep crisis. This study was set out to investigate molecular mechanisms of action of new iodine-containing nano-micelle drug FS-1, which potentially may improve the antibiotic therapy of drug resistant infections. Iodine is one of the oldest antimicrobials and until now there were no reports on development of resistance to iodine. Recent studies showed promising results on application of iodine-containing nano-micelles against antibiotic resistant pathogens as a supplement to antibiotic therapy. The mechanisms of action, however, remain unclear. The collection strain Escherichia coli ATCC BAA-196 showing an extended spectrum of resistance to beta-lactam and aminoglycoside antibiotics was used in this study as a model organism. Antibiotic resistance patterns, whole genomes and total RNA sequences of the FS-1 treated (FS) and negative control (NC) variants of E. coli BAA-196 were obtained and analyzed. FS culture showed an increased susceptibility to antibiotics associated with profound gene expression alterations switching the bacterial metabolism to anaerobic respiration, increased anabolism, osmotic stress response and inhibition of many nutrient uptake systems. Nucleotide methylation pattern were identified in FS and NC cultures. While the numbers of methylated sites in both genomes remained similar, some peculiar alterations were observed in their distribution along chromosomal and plasmid sequences.

Antimicrobial peptides (AMPs) have gained attention recently due to increasing antibiotic resistance amongst pathogens. Most AMPs are cationic in nature and their preliminary interactions with the negatively charged cell surface is mediated by electrostatic attraction. This is followed by pore formation, which is either receptor-dependent or -independent and leads to cell death. Typically, AMPs are characterized by their killing activity using bioactivity assays to determine host range and degree of killing. However, cell surface binding is independent from killing. Most of the studies performed to-date have attempted to quantify the peptide binding using artificial membranes. Here, we use the narrow-spectrum class IIa bacteriocin AMP pediocin PA-1 conjugated to a fluorescent dye as a probe to monitor cell surface binding. We developed a flow cytometry-based assay to quantify the strength of binding in target and non-target species. Through our binding assays, we found a strong positive correlation between cell surface charge and pediocin PA-1 binding. Interestingly, we also found inverse correlation between zeta potential and pediocin PA-1 binding, the correlation coefficient for which improved when only Gram-positives were considered. We also show the effect of the presence of protein, salt, polycationic species, and other non-target species on the binding of pediocin PA-1 to the target organism. We conclude that the of presence of highly charged non-target species, as well as solutes, can decrease the binding, and the apparent potency, of pediocin PA-1. Thus, these outcomes are highly significant to the use of pediocin PA-1 and related AMPs in mixed microbial settings such as those found in the gut microbiota.

Rifaximin is a low solubility antibiotic with activity against a wide range of bacterial pathogens. It accumulates in the intestine and is suitable for prolonged use. Three chemostat models (A, B and C) were used to investigate the effects of three rifaximin formulations (, {beta} and {kappa}, respectively) on the gut microbiome. Bacterial populations were monitored by bacterial culture and 16S rRNA gene amplicon (16S) sequencing. Limited disruption of bacterial populations was observed for rifaximin , {beta} and {kappa}. All formulations caused declines in total spores ([~]2 log10 cfu ml-1), Enterococcus spp. ([~]2 log10 cfu ml-1 in models A and C, and [~]1 log10 cfu ml-1 in model B), and Bacteroides spp. populations ([~]3 log10 cfu ml-1 in models A and C, and [~]4 log10 cfu ml-1 in model B). Bacterial populations fully recovered during antibiotic dosing in model C, and before the end of the experiment in models A and B. According to the taxonomic analysis, prior to rifaximin exposure, Bifidobacteriaceae, Ruminococcaceae, Acidaminococcaceae, Lachnospiraceae and Rikenellaceae families represented >92% of the total relative abundance, in all models. Within these families, 15 bacterial genera represented >99% of the overall relative abundance. Overall, the 16S sequencing and culture data showed similar variations in the bacterial populations studied. Among the three formulations, rifaximin {kappa} appeared to have the least disruptive effect on the colonic microbiota, with culture populations showing recovery in a shorter period and the taxonomic analysis revealing the least global variation in relative abundance of prevalent groups.

Bacteriophages are an increasingly attractive option for the treatment of antibiotic resistant infections, but their efficacy is difficult to discern due to confounding effects of antibiotics. Phages are generally delivered in conjunction with antibiotics, and thus, when patients improve its unclear whether the phages, antibiotics or both are responsible. This question is particularly relevant for enterococcus infections, as limited data suggest phages might restore antibiotic efficacy against resistant strains. Enterococci can develop high-level resistance to vancomycin, a primary treatment. We assessed clinical and laboratory isolates of Enterococcus faecium and Enterococcus faecalis to determine whether we could observe synergistic interactions between phages and antibiotics. We identified synergy between multiple phages and antibiotics including linezolid, ampicillin, and vancomycin. Notably, antibiotic susceptibility did not predict synergistic interactions with phages. Vancomycin resistant isolates (n=6) were eradicated by the vancomycin-phage combination as effectively as vancomycin susceptible isolates (n=2). Transcriptome analysis revealed significant gene expression changes under antibiotic-phage conditions, especially for linezolid and vancomycin, with upregulated genes involved in nucleotide and protein biosynthesis and downregulated stress response and prophage-related genes. While our results do not conclusively determine the etiology of the observed synergistic interactions between antibiotics and phages, they do confirm and build upon previous research that observed these synergistic interactions. Our work highlights how using phages can restore the effectiveness of vancomycin against resistant isolates. This finding provides a promising, although unexpected, strategy for moving forward with phage treatments for Vancomycin Resistant Enterococcus infections.

Clostridioides difficile infections are a growing concern in the hospital setting. Current prevention methods include infection control strategies, antibiotic stewardship, and proper hand hygiene. However, the occurrence of C. difficile still manages to cause nosocomial outbreaks especially in vulnerable populations. Probiotics have been historically questioned for their use in the prevention of antibiotic-associated diarrhea and more specifically, C. difficile infections. This meta-analysis pools 10 randomized controlled trials for the prevention of Clostridioides difficile infections (CDI) from reviewing the Cochrane Central Register of Controlled Trials (CENTRAL). A priori inclusion criteria remained as follows: RCTs, blinded/open trials, all populations, articles, or conference abstracts. Exclusion criteria excluded publications published outside 2013-2023*, non-English language trials, pre-clinical trials/protocols, case reports/series/systematic reviews, duplicates, probiotics not specified in methods, non-RCTs, incomplete/no outcomes reported, no confirmation of Clostridioides difficile infection. Probiotic strains tested in these trials mainly included Lactobacillus spp. and Bifidobacterium spp. Some studies showed significant benefits while others did not find significant improvement in the prevention of C. difficile infections. The meta-analysis suggests that probiotics may have a positive effect in preventing CDI during antibiotic treatment. The study results had large levels of statistical heterogeneity which indicates an argument for further large-scale research to provide more definitive evidence on the efficacy of probiotics in CDI prevention.

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.

BackgroundColistin is one of the last-line antimicrobial agents against drug-resistant gram-negative bacteria. Currently, little is known about the genetic mechanisms underlying colistin resistance in Shewanella algae, partly due to complex epistatic interactions among multiple genes. Methodology/Principal FindingsThis study sequenced, assembled, and compared the genomes of 23 mcr-negative colistin-resistant Shewanella algae from marine, clam, oyster, and human. Comparative genomics and computational approach were applied to find combinatorial mutations. A combination of three mutations (PmrB451, PmrE168, PmrH292) was found to be strongly associated with colistin resistance in Shewanella algae. Conclusions/SignificanceThis study demonstrates a computational approach for identifying epistatic-interacted mutations. Author summaryShewanella algae is an emerging pathogen related to Neglected Tropical Diseases (NTDs), including cobra-bite wound infections, marine injuries or ingestion of contaminated seafood. Shewanella algae is intrinsic resistant to various classes of {beta}-lactams. Additionally, growing resistance to colistin in mcr-negative Shewanella algae further limits therapeutic options, especially in resource-limited regions. Currently, little is known about the genetic mechanisms underlying colistin resistance in Shewanella algae, partly due to complex epistatic interactions among multiple genes. We conduct comparative genomics to identify combinatorial colistin resistance mutations in mcr-negative colistin-resistant Shewanella algae and a combination of three mutations (PmrB451, PmrE168, PmrH292) is strongly associated with colistin-resistance.

The gut microbiota is an immense reservoir of antimicrobial resistance genes (ARGs); however, in Australia the profile of the gut resistome, or ensemble of ARGs, has not been investigated. This study provides a first preliminary mapping of the major bacterial ARGs present in human, domestic dog and wild duck fecal samples collected from south-eastern Victoria, Australia; and evaluates the use of shotgun metagenomics sequencing (SMS) and targeted amplification of ARGs. We analysed SMS data using an in-house method and web-based bioinformatics tools: ResFinder and KmerResistance. We examined targeted sequences using One Codex or the PanBacterialAnalysis Torrent Suite plugin. All methods detected ARGs in all samples, with resistance to up to 13 classes of antibiotics detected overall. ARGs were more abundant in the human and dog samples than the duck samples. They mostly conferred resistance to three classes of antibiotics that are the most frequently prescribed in Australia: tetracycline, {beta}-lactams and MLSB (macrolide, lincosamide, streptogramin B). Targeted sequencing significantly improved sensitivity for detection of ARGs included in the panel; however, SMS provided quantitative information and allowed tentative identification of the host bacteria. For SMS, web-based and in-house methods gave comparable results, with discrepancies mostly due to different reference databases. The in-house method allowed manually checking results and potential errors, while web-based methods were user-friendlier and less time-consuming. More samples need to be investigated to fully describe the resistome in humans and animals in Australia.

SynopsisO_ST_ABSBackgroundC_ST_ABSThe methods to evaluate the interactions between Phages and antibacterials are unclear. As the laboratory methodologies used to assess conventional antibacterials are well established, we asseassed their efficacy in evaluating phage plus antibacterial. Methods100 multidrug resistant E. coli strains were used with three previously isolated and characterised E. coli phages of known efficacy. These phages UP17, JK08, 113 were assessed both individually and in a 1:1:1cocktail. In a Phage Microbial Inhibitory Concentration (PmIC) assay, a range of phage concentrations from 101 -108 were inoculated with 5x105/well bacteria in microtitre plates. The first lysed, clear well was taken as the PmIC. Amikacin(AMI) and meropenem(MERO) MICs were determined by microbroth dilution methods(ISO 2776-1:2019) and in combination AMI and MERO MICs were measured with a fixed Phage concentration of 105/well. MICs were performed in triplicate. Time-Kill curves(TKC) were conducted at fosfomycin concentrations of 133, 50 and 5mg/L with and without phage. ResultsThe PmIC50/90 for UP17 were >108/>108; JK08 107/>108; 113 107/>108 and the 1:1:1 cocktail 106/>108. AMI MIC50/90 were 0.5/>16 and MERO 0.12/>16mg/L. The addition of UP17 to AMI increased AMI MICs >2 fold in 78 strains. Equivalent increases in AMI MIC were seen with 39 strains with JK08, 54 strains with 113 and 45 strains with the cocktails. In contrast, meropenem MICs in the presence of phage were reduced >2 fold in 24 strains with UP17. Equivalent decreases in MERO MIC were seen with 34 strains with JK08, 26 strains with 113 and 29 strains with the cocktails. In TKCs addition of phage suppressed regrowth. ConclusionMicrobroth methodologies based on ISO 2776-1:2019 and TKCs allow the interaction between Phages and antibacterials to be studied. Optimisation may produce laboratory-based methods with translational value.

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.

BackgroundAntimicrobial resistant (AMR) organisms in environment may harm people. This study assessed the phenotypic and genotypic characteristics of AMR organisms from drinking and wastewater. Materials and methodsThis cross-sectional study conducted randomly on 30 samples (15 drinking water samples from household places; 15 sewage lifts stations) and collected aseptically, filtered, inoculated and isolated from culture plates, identified biochemically of pathogenic bacteria, and disc diffusion tested for antibiotic susceptibility. The primers of the targeted antimicrobial resistance genes were used for molecular amplification. ResultsTwenty-five bacteria were isolated from 30 drinking and wastewater samples. Pseudomonas spp. (36%), Enterobacter spp. (28%), Escherichia coli (20%), Citrobacter spp.(4%), Acinetobacter spp (4%) and Klebsiella oxytoca (4%) were isolated. Most of the isolates exhibited resistance to multiple groups of antibiotics, with meropenem, imipenem, gentamicin, ciprofloxacin, and amikacin showing the highest sensitivity against the isolates. Multiplex PCR confirmed the presence of two ESBL genes (blaSHV and blaCTX-M-15) and five carbapenemase genes (blaIMP, blaVIM, blaKPC, blaOXA1, blaNDM1) in resistant bacteria and blaCTX-M-15 (53.3%) followed by blaKPC (46.7%) genes were the most prevalent from drinking and wastewater samples respectively. According to WHOs sanitary inspection risk score classification, 60% of drinking water samples scored 4 out of 11, categorizing them as intermediate risk based on hazard score. ConclusionsThis study addresses antimicrobial resistance in the environment, emphasizing public health implications; advocating for improved environmental regulations to mitigate AMR organism discharge through wastewater and drinking water. The significance of the studyThe study attempted to determine the pattern of antimicrobial resistance of microorganisms using phenotypic and genotypic methods by polymerase chain reaction (PCR), targeting particular genes with specific sequence of primers. As in Bangladesh very few studies for antimicrobial resistance organisms from drinking water and wastewater around household and hospital environments in Dhaka city, yet finished to change public health perspectives, and inform respective authorities for making decision. Thus the research contributes to generating some evidence-based information about the reservoir of antimicrobial resistance in environment.

Clostridioides difficile is an anaerobic, spore-forming, Gram-positive pathogen responsible for various conditions from mild diarrhea to severe toxic megacolon and potentially death. Current treatments for C. difficile infection (CDI) rely on antibiotics like vancomycin and metronidazole (MTZ); however, the high doses required often disrupt gut microbiota, leading to a high recurrence rate. Mitomycin C (MMC), a chemotherapy drug approved by the FDA, is known for inducing phage production in lysogenic bacterial strains, effectively targeting the host bacteria. Given that 70% of C. difficile strains harbor prophages, this study investigates MMCs potential to enhance antibiotic efficacy against CDI. Our in vitro experiments indicate that MMC acts synergistically with MTZ to inhibit the growth of C. difficile strain R20291. Furthermore, this combination decreases biofilm-resident vegetative cell resistance and reduces the MTZ concentration needed to kill C. difficile in stool samples ex vivo. In a CDI mouse relapse model, in vivo results show that MMC combined with a low dose of MTZ significantly improves survival rates and reduces fecal spore counts after antibiotic treatment. Overall, these findings suggest that a low-dose combination of MMC and MTZ offers enhanced therapeutic efficacy for CDI, both in vitro and in vivo, and may provide a promising new approach for treatment.

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.

AimsBacteriophage (phage) propagation has traditionally relied on bacterial culture media containing animal-derived ingredients; however, safety concerns with animal-derived materials for production of phages for therapeutic use limit their acceptability. We compared animal-free and traditional media formulations, and evaluated their effects on phage yield, bactericidal activity, and genomic characteristics, hypothesizing no significant differences would be observed. Methods and ResultsPhages targeting Pseudomonas aeruginosa (n=8) and Staphylococcus aureus (n=1) were propagated in solid and liquid media containing animal-free (AF) or animal-derived (LB) peptones. Kinetic assays were used to assess phage suppression of host bacterial growth. In a mock therapeutic phage screen, spot tests, Efficiency of Plating (EOP) and kinetic assays were performed against novel bacterial targets. Whole genome sequencing of phages and their bacterial hosts propagated in AF or LB broth was used to observe genomic differences between formulations. Animal-free peptone did not impact phage yield, with both AF and LB phage stocks growing to high titers ([&ge;]108 PFU/mL). Kinetic assay results showed similar suppression indices for AF and LB-grown phages. Likewise, phage screen spot test, EOP, and kinetic assay results were similar between AF and LB phages. Comparisons of phage and bacterial genome annotations showed no major differences arising from media formulation. ConclusionsFindings suggest animal-free peptones do not significantly alter phage yield, bactericidal activity, or genomic characteristics, supporting use of animal-free medium for medicinal phage manufacture. This is one of the first studies to systematically combine phenotypic and genomic assessment of phages and hosts across animal-free and traditional media. Impact StatementPhage therapy is increasingly used to treat antimicrobial resistance infections. Emerging guidelines and regulations for the manufacture of phage therapeutics will impact laboratory processes and materials used for phage production. Here, we explored the use of an animal-free medium for medicinal phage propagation, providing data on phage yield and metrics of phage activity.