Spread Of Drug Resistance Research Articles

Transcriptomic Analysis of the Effect of Glabridin on Biofilm Formation in Staphylococcus Aureus.

Staphylococcus aureus (S. aureus) is among the major skin infection-causing pathogens in animals and humans. Its ability to form biofilms has become a foremost cause of bacterial infections and the extensive spread of drug resistance, which poses a great difficulty in clinical treatment. Glabridin (Glb), an extract of licorice with antibacterial and anti-infective properties, has a partially understood biofilm-inhibitory mechanism. This study investigated the inhibitory and antibiofilm activities of subinhibitory concentrations of Glb against S. aureus. The crystal violet assay revealed that Glb significantly suppressed biofilm expression. Scanning electron microscopy observations unveiled that Glb reduced S. aureus adhesion and accumulation by disrupting the spatial structure of the biofilm. In vitro extracellular DNA (eDNA) inhibition assays demonstrated that Glb inhibited biofilm formation by S. aureus by suppressing eDNA secretion. In total, 184 differentially expressed genes were obtained through transcriptomic (RNA-seq) sequencing, of which 81 and 103 genes were upregulated and downregulated, respectively. Glb regulated the transcript levels of biofilm-related genes through the phosphatase transfer system, two-component regulatory system, and nitrogen metabolism. The qPCR analysis was performed to confirm whether Glb interfered with the expression of regulatory genes involved in S. aureus biofilm formation (SarA, ArlR, FnbA, ClfA, icaD, and icaR) as well as the virulence gene Hla. In conclusion, this study demonstrates that Glb has a significant inhibitory effect on biofilm activity and is expected to be a good antibiofilm drug.

PRIVATE SECTOR INVOLVEMENT IN TUBERCULOSIS CONTROL IN A TRIBAL DISTRICT OF MAHARASHTRA, INDIA

Introduction: In India, the private sector caters about seventy percent of TB patients. The majority of private practitioners (PPs) are not updated with current National Tuberculosis Elimination Program (NTEP) guidelines resulting in wrong diagnosis and treatment leading to irrational use of the anti-tuberculosis drugs favoring the emergence and spread of drug resistance. Objectives: 1)To assess the knowledge of PPs about diagnosis and management of tuberculosis as per NTEP guidelines 2)To know the difficulties faced by PPs during the treatment of TB patients and measures to address them as per their perspective. Methodology: Study design & study setting:- a cross-sectional study was undertaken in a tribal district of Maharashtra. 86 PPs interviewed with the help of a structured questionnaire and later conducted focused group discussion (FGD). The data was analyzed with percentages and proportions. Results: The majority 65(75%) of PPs had knowledge about NTEP guidelines but only 21(25%) PPs were updated with the latest changes in the program. PPs expressed concern over the non-availability of diagnostics for extra-pulmonary TB, occasional shortage of drugs, unawareness of patients about the gravity of the disease, and treatment compliance. To address it, they felt the need for awareness activities on social media continuously, round-the-clock services for investigations, incentive for successfully completing treatment to patients along with timely updating PPs about recent updates on NTEP. Conclusion: Continuous quality training regarding basic knowledge and recent updates in NTEP for PPs should be imparted NTEP implementation at the district needs to be strengthened to overcome the difficulties mentioned by PPs.

Risk of selection and timelines for the continued spread of artemisinin and partner drug resistance in Africa.

The introduction of artemisinin combination therapies (ACTs) has significantly reduced the burden of Plasmodium falciparum malaria, yet the emergence of artemisinin partial resistance (ART-R) as well as partner drug resistance threatens these gains. Recent confirmations of prevalent de novo ART-R mutations in Africa, in particular in Rwanda, Uganda and Ethiopia, underscore the urgency of addressing this issue in Africa. Our objective is to characterise this evolving resistance landscape in Africa and understand the speed with which ART-R will continue to spread. We produce estimates of both ART-R and partner drug resistance by bringing together WHO, WWARN and MalariaGen Pf7k data on antimalarial resistance in combination with a literature review. We integrate these estimates within a mathematical modelling approach, aincorporating to estimate parameters known to impact the selection of ART-R for each malaria-endemic country and explore scenarios of ART-R spread and establishment. We identify 16 malaria-endemic countries in Africa to prioritise for surveillance and future deployment of alternative antimalarial strategies, based on ART-R reaching greater than 10% prevalence by 2040 under current malaria burden and effective-treatment coverage. If resistance continues to spread at current rates with no change in drug policy, we predict that partner drug resistance will emerge and the mean percentage of treatment failure across Africa will reach 30.74% by 2060 (parameter uncertainty range: 24.98% - 34.54%). This translates to an alarming number of treatment failures, with 52,980,600 absolute cases of treatment failure predicted in 2060 in Africa (parameter uncertainty range: 26,374,200 - 93,672,400) based on current effective treatment coverage. Our results provide a refined and updated prediction model for the emergence of ART-R to help guide antimalarial policy and prioritise future surveillance efforts and innovation in Africa. These results put into stark context the speed with which antimalarial resistance may spread in Africa if left unchecked, confirming the need for swift and decisive action in formulating antimalarial treatment policies focused on furthering malaria control and containing antimalarial resistance in Africa. The rise of artemisinin partial resistance (ART-R) and increasing partner drug tolerance by Plasmodium falciparum malaria in Africa threatens to undo malaria control efforts. Recent confirmations of de novo ART-R markers in Rwanda, Uganda, and Ethiopia highlight the urgent need to address this threat in Africa, where the vast majority of cases and deaths occur. This study characterises the resistance landscape and predicts the spread of antimalarial resistance across Africa. We estimate and map the current levels of resistance markers related to artemisinin and its partner drugs using WHO, WWARN, and MalariaGen Pf7k data. We combine these estimates with current malaria transmission and treatment data and use an established individual-based model of malaria resistance to simulate future resistance spread. We identify 16 African countries at highest risk of ART-R for prioritisation of enhanced surveillance and alternative antimalarial strategies. We project that, without policy changes, ART-R will exceed 10% in these regions by 2040. By 2060, if resistance spreads unchecked, we predict mean treatment failure rates will reach 30.74% (parameter uncertainty range: 24.98% - 34.54%) across Africa. This alarming spread of resistance is predicted to cause 52.98 million treatment failures (uncertainty range: 26.37 million - 93.67 million) in 2060. The impact of antimalarial resistance in Africa, if left unchecked, would hugely damage efforts to reduce malaria burden. Our results underscore the critical need for swift policy action to contain resistance and guide future surveillance and intervention efforts.

ResMAP-a saturation mutagenesis platform enabling parallel profiling of target-specific resistance-conferring mutations in Plasmodium.

An increase in treatment failures for malaria highlights an urgent need for new tools to understand and minimize the spread of drug resistance. We describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium spp, the causative agent of malaria. Saturation mutagenesis was used to generate a mutation library containing all conceivable mutations for a region of the antimalarial-binding site of a promising drug target, Plasmodium falciparum lysyl tRNA synthetase (PfKRS). Screening of this high-coverage library with characterized PfKRS inhibitors revealed multiple resistance-conferring substitutions including several clinically relevant mutations. Genetic validation of these mutations confirmed resistance of up to 100-fold and computational modeling dissected their role in drug resistance. We discuss potential applications of this data including the potential to design compounds that can bypass the most serious resistance mutations and future resistance surveillance.

A Strategy of Killing Two Birds With One Stone for Blocking Drug Resistance Spread With Engineered Bdellovibrio bacteriovorus.

Drug-resistant pathogens significantly threaten human health and life. Simply killing drug-resistant pathogens cannot effectively eliminate their threat since the drug-resistant genes (DRGs) released from dead drug-resistant pathogens are difficult to eliminate and can further spread via horizontal gene transfer, leading to the spread of drug resistance. The development of antibacterial materials with sterilization and DRGs cleavage activities is highly crucial. Herein, a living system, Ce-PEA@Bdello, is fabricated with bacterial killing and DRGs cleavage activities for blocking bacterial drug resistance dissemination by engineered Bdellovibrio bacteriovorus (Bdello). Ce-PEA@Bdello is obtained by engineering Bdello with dopamine and a multinuclear cerium (IV) complex. Ce-PEA@Bdello can penetrate and eliminate kanamycin-resistant P. aeruginosa (KanR) biofilms via the synergistic effect of predatory Bdello and photothermal polydopamine under near-infrared light. Additionally, the DNase-mimicking ability of Ce-PEA@Bdello endows it with genome and plasmid DNA cleavage ability. An in vivo study reveals that Ce-PEA@Bdello can eliminate P. aeruginosa (KanR) and cleave DRGs in scald/burn infected wounds to block the spread of drug resistance and accelerate wound healing. This bioactive system constructed from natural living materials offers a promising means for blocking the spread of drug resistance.

Enhancing the Intrinsic Antiplasmodial Activity and Improving the Stability and Selectivity of a Tunable Peptide Scaffold Derived from Human Platelet Factor 4.

The control of malaria, a disease caused by Plasmodium parasites that kills over half a million people every year, is threatened by the continual emergence and spread of drug resistance. Therefore, new molecules with different mechanisms of action are needed in the antimalarial drug development pipeline. Peptides developed from host defense molecules are gaining traction as anti-infectives due to theood of inducing drug resistance. Human platelet factor 4 (PF4) has intrinsic activity against P. falciparum, and a macrocyclic helix-loop-helix peptide derived from its active domain recapitulates this activity. In this study, we used a stepwise approach to optimize first-generation PF4-derived internalization peptides (PDIPs) by producing analogues with substitutions to charged and hydrophobic amino acid residues or with modifications to terminal residues including backbone cyclization. We evaluated the in vitro activity of PDIP analogues against P. falciparum compared to their overall helical structure, resistance to breakdown by serum proteases, selective binding to negatively charged membranes, and hemolytic activity. Next, we combined antiplasmodial potency-enhancing substitutions that retained favorable membrane and cell-selective properties onto the most stable scaffold to produce a backbone cyclic PDIP analogue with four-fold improved activity against P. falciparum compared to first-generation peptides. These studies demonstrate the ability to modify PDIP to select for and combine desirable properties and further validate the suitability of this unique peptide scaffold for developing a new molecule class that is distinct from existing antimalarial drugs.

Phylogenetic Network Analyses Reveal the Influence of Transmission Clustering on the Spread of HIV Drug Resistance in Quebec from 2002 to 2022.

HIV drug resistance (HIV-DR) may jeopardize the benefit of antiretroviral therapy (ART) in treatment and prevention. This study utilized viral phylogenetics to resolve the influence of transmission networks on sustaining the spread of HIV-DR in Quebec spanning 2002 to 2022. Time trends in acquired (ADR) and transmitted drug resistance (TDR) were delineated in treatment-experienced (n = 3500) and ART-naïve persons (n = 6011) with subtype B infections. Similarly, non-B-subtype HIV-DR networks were assessed pre- (n = 1577) and post-ART experience (n = 488). Risks of acquisition of resistance-associated mutations (RAMs) were related to clustering using 1, 2-5, vs. 6+ members per cluster as categorical variables. Despite steady declines in treatment failure and ADR since 2007, rates of TDR among newly infected, ART-naive persons remained at 14% spanning the 2007-2011, 2012-2016, and 2017-2022 periods. Notably, half of new infections among men having sex with men and heterosexual groups were linked in large, clustered networks having a median of 35 (14-73 IQR) and 16 (9-26 IQR) members per cluster, respectively. Cluster membership and size were implicated in forward transmission of non-nucleoside reverse transcriptase inhibitor NNRTI RAMs (9%) and thymidine analogue mutations (TAMs) (5%). In contrast, transmission of M184V, K65R, and integrase inhibitors (1-2%) remained rare. Levels of TDR reflected viral replicative fitness. The median baseline viremia in ART-naïve groups having no RAMs, NNRTI RAMs, TAMs, and M184VI were 46.088, 38,447, 20,330, and 6811 copies/mL, respectively (p < 0.0001). Phylogenetics emphasize the need to prioritize ART and pre-exposure prophylaxis strategies to avert the expansion of transmission cascades of HIV-DR.

Antagonistic activity of monocultures and consortia of lactobacilli against multidrug-resistant isolates of opportunistic bacteria as a screening of their probiotic potential

Background. In recent years, special attention has been paid to the studying the consortia of probiotic bacteria. In these associations, the properties of individual microorganisms can be enhanced, in particular, their antagonistic activity which is an effective indicator for screening of probiotic potential. The development of probiotics based on such consortia with antibacterial properties is critical in the light of the growing problem of drug resistance in microorganisms.The aim of the work. To study the antagonistic activity of monocultures and consortia of lactobacilli against multidrug-resistant isolates of opportunistic bacteria. Materials and methods. The antagonistic activity of lactobacilli monocultures and their consortia was assessed simultaneously by two methods: the cross streak method and the well diffusion method.Results. All strains of lactobacilli and their consortia, depending on the research method, had varying degrees of antagonistic activity. Five consortia had stronger antagonism to test cultures as compared to monocultures, while in one consortium, the effect of antagonistic activity was reduced compared to monocultures. The results of studying the antagonistic activity of two consortia (Limosilactobacillus fermentum 44/1 and Lacticaseibacillus rhamnosus 12L, Latilactobacillus curvatus LCR-111-1 and Lactiplantibacillus plantarum 8PAZ) contradict data on the biocompatibility of strains in these consortia. Differences in the degree of antagonistic effects of lactobacilli on gram-positive and gram-negative species of opportunistic bacteria were revealed.Conclusion. The study showed that both the biocompatibility of the probiotic strains and the antagonistic activity of the consortium are the important requirements for creating a probiotic consortium with effective probiotic potential. To study the antagonistic properties of lactobacilli, the number of isolates of target gram-positive and gram-negative bacteria and normobiota should be increased. This will allow us to determine effective strategies for using probiotics in conditions of the spread of drug resistance of microorganisms.

A newly identified IncY plasmid from multi-drug-resistant Escherichia coli isolated from dairy cattle feces in Poland.

This study reveals the identification of new strains of antibiotic-resistant Escherichia coli in cattle manure from a dairy farm in Poland, offering critical insights into the spread of drug resistance. Through whole-genome sequencing, researchers discovered novel plasmids within these bacteria, which carry genes resistant to multiple antibiotics. These findings are particularly alarming, as these plasmids can transfer between different bacterial species, potentially escalating the spread of antibiotic resistance. This research underscores the vital connection between the health of humans, animals, and the environment, emphasizing the concept of One Health. It points to the critical need for global vigilance and strategies to curb the proliferation of antibiotic resistance. By showcasing the presence of these strains and their advanced resistance mechanisms, the study calls for enhanced surveillance and preventive actions in both agricultural practices and healthcare settings to address the imminent challenge of antibiotic-resistant bacteria.

Characterization and genetic analysis of extensively drug-resistant hospital acquired Pseudomonas aeruginosa isolates

BackgroundThe incidence of hospital-acquired infections in extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) has been increasing worldwide and is frequently associated with an increase in mortality and morbidity rates. The aim of this study was to characterize clinical XDR-PA isolates recovered during six months at three different hospitals in Egypt.ResultsSeventy hospital-acquired clinical isolates of P. aeruginosa were classified into multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR), according to their antimicrobial resistance profile. In addition, the possession of genes associated with mobile genetic elements and genes encoding antimicrobial resistance determinants among isolates were detected using polymerase chain reaction. As a result, a significant percentage of the isolates (75.7%) were XDR, while 18.5% were MDR, however only 5.7% of the isolates were non-MDR. The phenotypic detection of carbapenemases, extended-spectrum β-lactamases (ESBLs) and metallo β-lactamase (MBL) enzymes showed that 73.6% of XDR-PA isolates were carbapenemases producers, whereas 75.5% and 88.7% of XDR-PA isolates produced ESBLs and MBL respectively. In addition, PCR screening showed that oxa gene was the most frequently detected gene of carbapenemases (91.4%), while aac(6ʹ)-lb gene was mostly detected (84.3%) among the screened aminoglycosides-resistance genes. Furthermore, the molecular detection of the colistin resistance gene showed that 12.9% of isolates harbored mcr-1 gene. Concerning mobile genetic element markers (intI, traA, tnp513, and merA), intI was the highest detected gene as it was amplified in 67 isolates (95.7%). Finally, phylogenetic and molecular typing of the isolates via ERIC-PCR analysis revealed 10 different ERIC fingerprints.ConclusionThe present study revealed a high prevalence of XDR-PA in hospital settings which were resistant to a variety of antibiotics due to several mechanisms. In addition, 98% of the XDR-PA clinical isolates contained at least one gene associated with movable genetic elements, which could have aided the evolution of these XDR-PA strains. To reduce spread of drug resistance, judicious use of antimicrobial agents and strict infection control measures are therefore essential.

A Plasmodium falciparum genetic cross reveals the contributions of pfcrt and plasmepsin II/III to piperaquine drug resistance.

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin as a first-line treatment against malaria. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multidrug-resistant KEL1/PLA1 lineage isolate (KH004) and a drug-susceptible Malawian parasite (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and multiple copies of pm2/3. We recovered 104 unique recombinant parasites and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3. We performed a detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes with these progenies, including PPQ survival assay, area under the dose response curve, and a limited point IC50. We find that inheritance of the KH004 pfcrt allele is required for reduced PPQ sensitivity, whereas copy number variation in pm2/3 further decreases susceptibility but does not confer resistance in the absence of additional mutations in pfcrt. A deep investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background to a range of PPQ-related traits. Additionally, we find that the resistance phenotype associated with parasites inheriting the G367C substitution in pfcrt is consistent with previously validated PPQ resistance mutations in this transporter.IMPORTANCEResistance to piperaquine, used in combination with dihydroartemisinin, has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and has the distinct advantage of quantifying the effects of naturally evolved genetic variation. Our study was strengthened since the full range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of the pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multigene model suggests an important role for both loci in the evolution of this multidrug-resistant parasite lineage.

Comprehensive Overview of Candida auris: An Emerging Multidrug-Resistant Fungal Pathogen.

The rise of Candida auris, a multidrug-resistant fungal pathogen, across more than 40 countries, has signaled an alarming threat to global health due to its significant resistance to existing antifungal therapies. Characterized by its rapid spread and robust drug resistance, C. auris presents a critical challenge in managing infections, particularly in healthcare settings. With research on its biological traits and genetic basis of virulence and resistance still in the early stages, there is a pressing need for a concerted effort to understand and counteract this pathogen. This review synthesizes current knowledge on the epidemiology, biology, genetic manipulation, pathogenicity, diagnostics, and resistance mechanisms of C. auris, and discusses future directions in research and therapeutic development. By exploring the complexities surrounding C. auris, we aim to underscore the importance of advancing research to devise effective control and treatment strategies.

Inhibitory Potential of Bifidobacterium longum FB1-1 Cell-Free Supernatant against Carbapenem-Resistant Klebsiella pneumoniae Drug Resistance Spread.

The widespread dissemination of carbapenem-resistant Klebsiella pneumoniae (CRKP) and its drug resistance transfer poses a global public health threat. While previous studies outlined CRKP's drug resistance mechanism, there is limited research on strategies inhibiting CRKP drug resistance spread. This study investigates the potential of Bifidobacterium longum (B. longum) FB1-1, a probiotic, in curbing the spread of drug resistance among CRKP by evaluating its cell-free supernatant (CFS) for antibacterial activity. Evaluating the inhibitory effect of FB1-1 CFS on CRKP drug resistance spread involved analyzing its impact on drug resistance and virulence gene expression; drug resistance plasmid transfer FB1-1 CFS exhibited an MIC range of 125 μL/mL against CRKP. After eight hours of co-culture, CFS achieved a 96% and 100% sterilization rate at two and four times the MIC, respectively. At sub-inhibitory concentrations (1/2× MIC), FB1-1 CFS reduced the expression of the bla_KPC gene, which is pivotal for carbapenem resistance, by up to 62.13% across different CRKP strains. Additionally, it markedly suppressed the expression of the uge gene, a key virulence factor, by up to 91%, and the fim_H gene, essential for bacterial adhesion, by up to 53.4%. Our study primarily focuses on determining the inhibitory effect of FB1-1 CFS on CRKP strains harboring the bla_KPC gene, which is a critical resistance determinant in CRKP. Furthermore, FB1-1 CFS demonstrated the ability to inhibit the transfer of drug resistance plasmids among CRKP strains, thus limiting the horizontal spread of resistance genes. This study highlights FB1-1 CFS's inhibitory effect on CRKP drug resistance spread, particularly in strains carrying the bla_KPC gene, thus offering a novel idea and theoretical foundation for developing antibacterial drugs targeting CRKP resistance.

A Mathematical Analysis of Competitive Dynamics and Aggressive Treatment in the Evolution of Drug Resistance in Malaria Parasites

Experimental evidence supports the counterintuitive notion that rapid eradication of pathogens within a host, infected with both drug-sensitive and -resistant malaria parasites, can actually accelerate the evolution of drug-resistant pathogens. This study aims to analyze the competitive dynamics between these two strains through a mathematical model and evaluate the impact of aggressive treatment on the spread of drug resistance. We conducted equilibrium, uncertainty, and sensitivity analyses to assess the model, identifying and measuring the influence of key factors on the outcome variable (the population of drug-resistant parasites). Both equilibrium and local sensitivity analyses concurred that the density of drug-resistant parasites is notably affected by genetic instability, the production rate of red blood cells, the number of merozoites, and competition factors. Conversely, there is a negative relationship between genetic instability and one of the competition coefficients. Global sensitivity analysis offers a comprehensive examination of the impact of each input parameter on the temporal propagation of drug resistance, effectively accounting for the interplay among parameters. Both local and global sensitivity analyses underscore the continuous impact of drug treatment on the progression of drug resistance over time. This paper anticipates exploring the underlying mechanisms of drug resistance and providing theoretical support for developing more effective drug treatment strategies.

Cytotoxicity and Biological Activities of Geopopolis Extract from the Stingless bee (Melipona scutellaris) in Isolates of Staphylococcus aureus.

Geopropolis resins are produced by stingless bees (Meliponinae), developed from the collection of resinous materials, waxes and exudates, from the flora of the region where stingless bees are present, in addition to the addition of clay or earth in its composition. Several biological activities are attributed to Ethanol Extracts of Geopropolis (EEGP). The bioactive properties are associated with the complex chemical composition that the samples have. This work aims to evaluate the biological activities of the EEGP, in order to contribute with a natural therapeutic alternative, to face infections, mainly those caused by resistant strains of Staphylococcus aureus. The EEGP MIC tests showed antibacterial activity against two strains of S. aureus, both at concentrations of 550 μg/mL. The MBC performed with the inhibition values showed that the EEGP has bacteriostatic activity in both strains. Biofilm inhibition rates exhibited an average value greater than 65 % at the highest concentration. The EEGP antioxidant potential test showed good antioxidant activity (IC50) of 11.05±1.55 μg/mL. In the cytotoxicity test against HaCat cells, after 24 hours, EEGP induced cell viability at the three tested concentrations (550 μg/mL: 81.68±3.79 %; 1100 μg/mL: 67.10±3.76 %; 2200 μg/mL: 67.40±1.86 %). In view of the above, the safe use of EEGP from the brazilian northeast could be proven by the cytotoxicity test, and its use as an antioxidant and antibacterial agent has proven to be effective, as an alternative in combating oxidative stress and microorganisms such as S. aureus, which, through the spread and ongoing evolution of drug resistance, generates an active search for effective solutions.

93 Evaluating the efficacy of anthelmintics in an upper Midwest horse herd

Abstract Cyathostomins in the gastrointestinal tract are ubiquitous in horses around the world and remain a target in parasite control programs. Resistance to anthelmintics has risen in recent years with widespread resistance to benzimidazoles and pyrantels and emerging resistance to macrocyclic lactones. Thus, investigating the efficacy of anthelmintics in different locations is important to direct future parasite control programs. This 2-yr study evaluated the efficacy of three anthelmintic drugs in a university-owned equine herd in the upper Midwest using fecal egg count reduction tests (FECRT). Additionally, the effect of age group and body condition score (BCS) on fecal egg shedding was evaluated. We hypothesized that drug resistance is present in the parasite population of this equine herd. Fecal egg counts (FEC) of strongelid eggs were determined using the McMaster method. The efficacy of anthelmintics was determined by calculating the percentage of reduction in FEC between d 0 and d 14 post-treatment. Eighteen horses of various breeds were used in the study. The horses were grouped by age (&amp;lt; 10, 10-16, and &amp;gt; 16 yr; n = 6 in each group) and BCS using the Henneke system (5.0-5.5, n = 9; 6.0-6.5., n = 6; and 7.0-8.0, n = 3). In yr 1, horses within age group were randomly assigned to anthelmintic treatment: pyrantel pamoate, fenbendazole, or ivermectin. In yr 2, all horses received ivermectin. Individual FEC were performed on the day of anthelmintic treatment (d 0) using freshly voided feces. FEC were repeated 14 d after treatment. FEC were log transformed before analysis. Efficacy of the anthelmintics were compared using a mixed model ANOVA with repeated measures (SAS 9.4). Results were considered significant when P &amp;lt; 0.05. The FECRT was greater when treated with ivermectin and pyrantel pamoate (94.8 ± 11.6 % and 96.6 ± 14.3 %, respectively) compared with fenbendazole (54.4 ± 10.8 %; P &amp;lt; 0.05). The FECRT increased from 54.4% when treated with fenbendazole to 100% when the same horses were treated with ivermectin (P &amp;lt; 0.05). Age tended to affect egg shedding in horses (P = 0.0548). Young horses (&amp;lt; 10 yr) had a greater FEC compared with older horses (&amp;gt;16 yr; P &amp;lt; 0.05) but were not different from horses aged 10 to 16 yr (P &amp;gt; 0.05). There was no effect of BCS on strongelid egg shedding in this study (P = 0.9468). These results indicate the efficacy was within the expected range with no signs of resistance to pyrantel pamoate (93.3 to 100% reduction). There was evidence of emerging resistance to ivermectin (70.4 to 100% reduction) and significant resistance to fenbendazole (0 to 71.5% reduction) in the cyathostomin population in this horse herd. The findings of this study agree with previous research demonstrating resistance to anthelmintics. These results highlight the importance of designing improved parasite control programs to limit the spread of drug resistance with the ultimate goal of improving overall health of the horse.

Validation of deep amplicon sequencing of Dicrocoelium in small ruminants from Northern regions of Pakistan.

Dicrocoelium lancet flukes cause significant production loss in ruminant livestock. Although co-infection with multiple Dicrocoelium species within a host is common, techniques for studying the composition of these complex parasite communities are lacking. The pathogenicity, epidemiology, and therapeutic susceptibility of different helminth species vary, and little is known about the interactions that take place between co-infecting species and their hosts. Here, we describe the first applicationof metabarcoding deep amplicon sequencing method to studythe Dicrocoelium species in sheep and goats. First, rDNA ITS-2 sequences of four Dicrocoelium species (Dicrocoelium dendriticum, Dicrocoelium hospes, Dicrocoelium orientalis, and Dicrocoelium chinensis) were extracted from the NCBI public database. Phylogenetic analysis revealed separate clades of Dicrocoelium species; hence, molecular differentiation between each species is possible in co-infections. Second, 202 flukes belonging to seventeen host populations (morphologically verified as belonging to the Dicrocoelium genus) were evaluated to determine the deep amplicon sequencing read threshold of an individual fluke for each of the four species. The accuracy of the method in proportional quantification of samples collected from single hosts was further assessed. Overall, 198 (98.01%) flukes were confirmed as D. dendriticum and 1.98% produced no reads. The comparison of genetic distances between rDNA ITS-2 revealed 86% to 98% identity between the Dicrocoelium species. Phylogenetic analysis demonstrated a distinct clustering of species, apart from D. orientalis and D. chinensis, which sit very close to each other in a single large clade whereas D. hospes and D. dendriticum are separated into their own clade. In conclusion each sample was identified as D. dendriticum based on the proportion of MiSeq reads and validated the presence of this group of parasites in the Gilgit Baltistan and Khyber Pakhtunkhwa provinces of Pakistan. The metabarcoding deep amplicon sequencing technology and bioinformatics pathway have several potential applications, including species interactions during co-infections, identifying the host and geographical distribution of Dicrocoelium in livestock, drug therapy response evaluation and understanding of the emergence and spread of drug resistance.

Characterization of drug resistance genes in Indian Plasmodium falciparum and Plasmodium vivax field isolates

One of the major challenges for malaria control and elimination is the spread and emergence of antimalarial drug resistance. Mutations in Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) field isolates for five drug resistance genes viz. crt, mdr1, dhps, dhfr and kelch known to confer resistance to choloroquine (CQ), sulfadoxine-pyrimethamine (SP) and artemisinin (ART) and its derivatives were analyzed. A total of 342 symptomatic isolates of P. falciparum (Pf) and P. vivax (Pv) from 1993 to 2014 were retrieved from malaria parasite repository at National Institute of Malaria Research (NIMR). Sample DNA was extracted from dried blood spots and various targeted single nucleotide polymorphisms (SNPs) associated with antimalarial drug resistance were analysed for these isolates. 72S (67.7%) and 76T (83.8%) mutations along with SVMNT haplotype (67.7%) predominated the study population for Pfcrt. The most prevalent SNPs were 108N (73.2%) and 437G (24.8%) and the most prevalent haplotypes were ACNRNI (51.9%) and SAKAA (74.5%) in Pfdhfr and Pfdhps respectively. Only two mutations in Pfmdr1, 86Y (26.31%) and 184F (56.26%), were seen frequently in our study population. No mutations associated with Pfk13 were observed. For Pv, all the studied isolates showed two Pvdhps mutations, 383G and 553G, and two Pfdhfr mutations, 58R and 117N. Similarly, three mutations, viz. 958M, 908L and 1076L were found in Pvmdr1. No variations were observed in Pvcrt-o and Pvk12 genes. Overall, our study demonstrates an increase in mutations associated with SP resistance in both Pf and Pv, however, no single nucleotide polymorphisms (SNPs) associated with ART resistance have been observed for either species. Various SNPs associated with CQ resistance were seen in Pf; whereas only Pvmdr1 associated resistant SNPs were observed in Pv. Therefore, molecular characterization of drug resistance genes is essential for timely monitoring and prevention of malaria by identifying the circulating drug resistant parasites in the country.

Antimalarial drug resistance profiling of Plasmodium falciparum infections in India using Ion Torrent deep sequencing

IntroductionTracking the emergence and spread of antimalarial drug resistance is critical for supporting progress towards the control and eventual elimination of malaria in South Asia, especially India. Plasmodium falciparum has evolved resistance to virtually every antimalarial drug, and significant progress has been made to identify the molecular genetic mechanisms involved in the most common types of resistance.MethodsAn amplicon sequencing protocol was used for molecular surveillance of antimalarial drug resistance in a total of 158 patient isolates collected from December 2012 to September 2015 from three sites in south, west and east India: Tamil Nadu, Gujarat, and Odisha respectively. Five full length Plasmodium falciparum genes whose mutant proteins are implicated in antimalarial drug resistance were investigated: Pfcrt for chloroquine, Pfdhfr for pyrimethamine, Pfdhps for sulfadoxine, Pfk13 for artemisinin and Pfmdr1 for resistance to multiple antimalarial drugs.ResultsWe observed a high proportion of wild-type Pfcrt and Pfdhfr haplotypes from the P. falciparum-dominant site Rourkela, while mutant Pfcrt and Pfdhfr haplotypes were fixed at the P. vivax dominant sites Chennai and Nadiad. The wild-type Pfdhps haplotype was predominant across all study sites. We identified mutations in the propeller domain of Pfk13, although they are not associated with resistance to artemisinin. Finally, using samples taken from the same patient on day 2, day 7, and day 14 after artemisinin combination treatment, we were able to observe changes in allele frequency of drug resistance genes during the course of an infection.DiscussionThis is the first high-throughput deep sequencing study of five full-length P. falciparum genes in clinical isolates from three different study sites in India with varying transmission ecologies. Amplicon sequencing based on ion-torrent has the potential to track changes in the frequency of drug resistant alleles as a patient is undergoing drug therapy, and to identify new resistance mutations as they increase in frequency in the patient. This study showed possibility of whole gene sequencing, leads to in-depth molecular surveillance of multiple antimalarial resistant candidates and furthermore suggests investigations on reversal of resistant genotypes due to usage of artemisinin combination therapy in P. falciparum prevalent areas of the country.

Genotyping USA laboratory-maintained isolates and European clinical isolates of Dirofilaria immitis to assess macrocyclic lactone susceptibility or resistance at predictive SNP sites using droplet digital PCR

Dirofilaria immitis is a parasitic nematode that causes cardiovascular dirofilariosis ("heartworm disease”) primarily in canids. The principal approach for mitigating heartworm infection involves the use of macrocyclic lactone (ML) for prophylaxis. Recent research has substantiated the emergence of D. immitis displaying resistance to MLs in the USA. Numerous factors, such as the mobility of companion animals and competent vectors could impact the spread of drug resistance. Genomic analysis has unveiled that isolates resistant to ML exhibit unique genetic profiles when compared to their wild-type (susceptible) counterparts. Out of the ten single nucleotide polymorphism (SNP) markers validated in clinical samples of D. immitis from the USA, four have demonstrated their effectiveness in distinguishing between isolates with varying ML efficacy phenotypes. This study explores the potential of these confirmed SNPs for conducting surveillance studies. Genotypic analysis using SNP markers emerges as a valuable tool for carrying out surveys and evaluating individual clinical isolates. Two USA laboratory-maintained isolates (Berkeley, WildCat) and twenty-five random European clinical samples of either adult worms or microfilariae (mf) pools isolated from domestic dogs, were tested by droplet digital PCR (ddPCR)-based duplex assay. This approach elucidates genetic evidence pertaining to the development of drug resistance and provides baseline data on resistance related genotypes in Europe. The data on these clinical samples suggests genotypes consistent with the continued efficacy of ML treatment regimens in Europe. In addition, this assay can be significant in discriminating cases of drug-resistance from those possibly due to non-compliance to the recommended preventive protocols.