Isoliensinine from Cissampelos pariera rhizomes exhibits potential gametocytocidal and anti-malarial activities against Plasmodium falciparum clinical isolates.

BACKGROUND: The unmet demand for effective malaria transmission-blocking agents targeting the transmissible stages of Plasmodium necessitates intensive discovery efforts. In this study, a bioactive bisbenzylisoquinoline (BBIQ), isoliensinine, from Cissampelos pariera (Menispermaceae) rhizomes was identified and characterized for its anti-malarial activity. METHODS: Malaria SYBR Green I fluorescence assay was performed to evaluate the in vitro antimalarial activity against D6, Dd2, and F32-ART5 clones, and immediate ex vivo (IEV) susceptibility for 10 freshly collected P. falciparum isolates. To determine the speed- and stage-of-action of isoliensinine, an IC50 speed assay and morphological analyses were performed using synchronized Dd2 asexuals. Gametocytocidal activity against two culture-adapted gametocyte-producing clinical isolates was determined using microscopy readouts, with possible molecular targets and their binding affinities deduced in silico. RESULTS: Isoliensinine displayed a potent in vitro gametocytocidal activity at mean IC50gam values ranging between 0.41 and 0.69 µM for Plasmodium falciparum clinical isolates. The BBIQ compound also inhibited asexual replication at mean IC50Asexual of 2.17 µM, 2.22 µM, and 2.39 µM for D6, Dd2 and F32-ART5 respectively, targeting the late-trophozoite to schizont transition. Further characterization demonstrated a considerable immediate ex vivo potency against human clinical isolates at a geometric mean IC50IEV = 1.433 µM (95% CI 0.917-2.242). In silico analyses postulated a probable anti-malarial mechanism of action by high binding affinities for four mitotic division protein kinases; Pfnek1, Pfmap2, Pfclk1, and Pfclk4. Additionally, isoliensinine was predicted to possess an optimal pharmacokinetics profile and drug-likeness properties. CONCLUSION: These findings highlight considerable grounds for further exploration of isoliensinine as an amenable scaffold for malaria transmission-blocking chemistry and target validation.

Discovery of novel inhibitors of multidrug-resistant Acinetobacter baumannii.

The recent emergence of multidrug-resistant Acinetobacter baumannii strains and the non-efficacy of currently available antibiotics against such infections have led to an urgent need for the development of novel antibacterials. In an effort to address this problem, we have identified three novel inhibitors, namely, D5, D12 and D6 using in silico screening with a homology model of the outer membrane protein W2 (OmpW2) from A. baumannii, as the proposed new drug target. OmpW is an eight-stranded ß-barrel protein involved in the transport of hydrophobic molecules across the outer membrane and maintenance of homeostasis under cellular stress. The antimicrobial activities of compounds D5, D12 and D6 were evaluated against a panel of clinical isolates of A. baumannii strains. These compounds inhibited the growth of the strains with minimum inhibitory concentration (MIC) ranges of 1-32µg/mL. Time-kill kinetic studies with the highly virulent and multidrug-resistant strain, A. baumannii 5075, indicated that D6 exhibited the highest bactericidal activity asa≥3log10 CFU/mL (99.9%) reduction in colony count from the initial inoculum was observed after 30min incubation. D5 and D12 reduced at least 1log10 CFU/mL (90%) of the initial inoculum after 24h. In conclusion, these three lead inhibitors have provided two distinct chemical scaffolds for further analog design and optimizations, using chemical synthesis, to develop more potent inhibitors of the pathogen.

Release of luminal exosomes contributes to TLR4-mediated epithelial antimicrobial defense.

Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense.

Rapid detection and statistical differentiation of KPC gene variants in Gram-negative pathogens by use of high-resolution melting and ScreenClust analyses.

In the United States, the production of the Klebsiella pneumoniae carbapenemase (KPC) is an important mechanism of carbapenem resistance in Gram-negative pathogens. Infections with KPC-producing organisms are associated with increased morbidity and mortality; therefore, the rapid detection of KPC-producing pathogens is critical in patient care and infection control. We developed a real-time PCR assay complemented with traditional high-resolution melting (HRM) analysis, as well as statistically based genotyping, using the Rotor-Gene ScreenClust HRM software to both detect the presence of bla(KPC) and differentiate between KPC-2-like and KPC-3-like alleles. A total of 166 clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii with various ß-lactamase susceptibility patterns were tested in the validation of this assay; 66 of these organisms were known to produce the KPC ß-lactamase. The real-time PCR assay was able to detect the presence of bla(KPC) in all 66 of these clinical isolates (100% sensitivity and specificity). HRM analysis demonstrated that 26 had KPC-2-like melting peak temperatures, while 40 had KPC-3-like melting peak temperatures. Sequencing of 21 amplified products confirmed the melting peak results, with 9 isolates carrying bla(KPC-2) and 12 isolates carrying bla(KPC-3). This PCR/HRM assay can identify KPC-producing Gram-negative pathogens in as little as 3 h after isolation of pure colonies and does not require post-PCR sample manipulation for HRM analysis, and ScreenClust analysis easily distinguishes bla(KPC-2-like) and bla(KPC-3-like) alleles. Therefore, this assay is a rapid method to identify the presence of bla(KPC) enzymes in Gram-negative pathogens that can be easily integrated into busy clinical microbiology laboratories.

Effect of drug treatment options on the mobility and expression of blaKPC.

OBJECTIVES: Both transposition and increases in gene expression have been implicated in the success of KPC-producing pathogens, but the stimulus required for these phenomena are unknown. It is possible that exposure to antimicrobials during patient treatment increases bla(KPC) expression or induces Tn4401 transposition. The purpose of this study was to determine if exposure to carbapenems or other antimicrobial drug classes could stimulate expression of bla(KPC) or the in vitro transposition of Tn4401. METHODS: Five KPC-producing clinical isolates were evaluated in this study. Gene expression of RNA from each isolate exposed to subinhibitory, MIC or suprainhibitory levels of antibiotics was evaluated using real-time RT-PCR. Southern blots were performed on plasmids from isolates exposed to subinhibitory levels of antibiotics. RESULTS: There were subtle changes in bla(KPC) RNA expression following antibiotic exposure that were both strain and drug dependent. Multiple plasmids ranging from ~8 to >200 kb were observed for the Enterobacteriaceae isolates, whereas the Pseudomonas aeruginosa isolate had one ~55 kb plasmid. No changes in hybridization patterns or binding intensity for the bla(KPC) probe were observed after antibiotic exposure. CONCLUSIONS: While the changes in bla(KPC) RNA expression are subtle, the different responses observed suggest both strain- and genera-specific variations in response to different antibiotic treatments.

Therapeutic response to artemisinin combination therapies among individuals with Plasmodium falciparum single infection vs mixed Plasmodium species infections: a retrospective posthoc analysis in Kisumu County, western Kenya.

OBJECTIVES: This study examined the treatment response of mixed vs single-species Plasmodium falciparum infections to artemisinin-based combination therapies (ACTs). METHODS: A total of 1211 blood samples collected on days 0, 7, 14, 21, 28, 35, and 42 from 173 individuals enrolled in two randomized ACT efficacy studies were tested for malaria using 18s ribosomal RNA-based real-time polymerase chain reaction. All recurrent parasitemia were characterized for Plasmodium species composition and time to reinfection during 42-day follow-up compared across ACTs. RESULTS: Day 0 samples had 71.1% (116/163) single P. falciparum infections and 28.2% (46/163) coinfections. A total of 54.0% (88/163) of individuals tested positive for Plasmodium at least once between days 7-42. A total of 19.3% (17/88) of individuals with recurrent infections were infected with a different Plasmodium species than observed at day 0, with 76.5% (13/17) of these "hidden" infections appearing after clearing P. falciparum present at day 0. Artesunate-mefloquine (16.4 hours) and dihydroartemisinin-piperaquine (17.6 hours) had increased clearance rates over artemether-lumefantrine (21.0 hours). Dihydroartemisinin-piperaquine exhibited the longest duration of reinfection prophylaxis. Cure rates were comparable across each species composition. CONCLUSION: No differences in clearance rates were found depending on whether the infection contained species other than P. falciparum. Significantly longer durations of protection were observed for individuals treated with dihydroartemisinin-piperaquine.

bla(KPC) RNA expression correlates with two transcriptional start sites but not always with gene copy number in four genera of Gram-negative pathogens.

Klebsiella pneumoniae carbapenemase (KPC)-producing organisms are therapeutically and diagnostically challenging. It is possible that bla(KPC) gene expression plays a role in the variability observed in clinical susceptibility testing. bla(KPC) transformants together with 10 clinical isolates representing four genera were evaluated for bla(KPC) copy number and gene expression and correlated with ß-lactam MIC data. The data suggest that mechanisms other than gene copy number and expression of bla(KPC) contribute to variability in susceptibility when testing KPC-producing isolates.

Synergism in Antiplasmodial Activities of Artemether and Lumefantrine in Combination with Securidaca longipedunculata Fresen (Polygalaceae).

Malaria is the most lethal parasitic disease in the world. The frequent emergence of resistance by malaria parasites to any drug is the hallmark of sustained malaria burden. Since the deployment of artemisinin-based combination therapies (ACTs) it is clear that for a sustained fight against malaria, drug combination is one of the strategies toward malaria elimination. In Sub-Saharan Africa where malaria prevalence is the highest, the identification of plants with a novel mechanism of action that is devoid of cross-resistance is a feasible strategy in drug combination therapy. Thus, artemether and lumefantrine were separately combined and tested with extracts of Securidaca longipedunculata, a plant widely used to treat malaria, at fixed extract-drug ratios of 4:1, 3:1, 1:1, 1:2, 1:3, and 1:4. These combinations were tested for antiplasmodial activity against three strains of Plasmodium falciparum (W2, D6, and DD2), and seven field isolates that were characterized for molecular and ex vivo drug resistance profiles. The mean sum of fifty-percent fractional inhibition concentration (FIC50) of each combination and singly was determined. Synergism was observed across all fixed doses when roots extracts were combined with artemether against D6 strain (FIC50 0.403 ± 0.068) and stems extract combined with lumefantrine against DD2 strain (FIC50 0.376 ± 0.096) as well as field isolates (FIC50 0.656 ± 0.067). Similarly, synergism was observed in all ratios when leaves extract were combined with lumefantrine against W2 strain (FIC50 0.456 ± 0.165). Synergism was observed in most combinations indicating the potential use of S. longipedunculata in combination with artemether and lumefantrine in combating resistance.

Antimicrobial Stewardship Challenges in the Deployed Setting.

INTRODUCTION: Up to 34% of combat trauma injuries are complicated by infection with multidrug-resistant organisms. Overutilization of antibiotics has been linked to increased multidrug-resistant organisms in combat-injured patients. Antimicrobial stewardship efforts at deployed medical treatment facilities have been intermittently reported; however; a comprehensive assessment of antimicrobial stewardship practices has not been performed. MATERIALS AND METHODS: A survey tool was modified to include detailed questions on antimicrobial stewardship practices at medical treatment facilities. A Joint Service, multidisciplinary team conducted on-site assessments and interviews to assess the status of antimicrobial stewardship best practices, with particular emphasis on antibiotic prophylaxis in combat injured, in the U.S. Central Command operational theaters. Limitations to implementing stewardship to the national standards were explored thematically. RESULTS: Nine Role 1, 2, and 3 medical facilities representing the range of care were assessed on-site. A total of 67% of the sites reported a formal antimicrobial stewardship program and 56% of the sites had an assigned head of antimicrobial stewardship. No military personnel in theater received training on antimicrobial stewardship and laboratory assets were limited. Personnel at these sites largely had access to Joint Trauma System guidelines describing antimicrobial prophylaxis for combat injured (89%), yet infrequently received feedback on their implementation and adherence to these guidelines (11%). CONCLUSIONS: Antimicrobial stewardship programs in theater are in the early stages of development in theater. Areas identified for improvement are access to expertise, development of a focus on high-impact lines of effort, laboratory support, and the culture of antimicrobial prescribing. Risks can be mitigated through theater level formalization of efforts, expert mentoring through telehealth, and a focus on implementation and adherence and feedback to national guidelines.

Status Update on Infection Prevention and Control at Deployed Medical Treatment Facilities.

INTRODUCTION: Infections with multidrug resistant organisms that spread through nosocomial transmission complicate the care of combat casualties. Missions conducted to review infection prevention and control (IPC) practices at deployed medical treatment facilities (MTFs) previously showed gaps in best practices and saw success with targeted interventions. An IPC review has not been conducted since 2012. Recently, an IPC review was requested in response to an outbreak of multidrug resistant organisms at a deployed facility. MATERIALS AND METHODS: A Joint Service team conducted onsite IPC reviews of MTFs in the U.S. Central Command area of operations. Self-assessments were completed by MTF personnel in anticipation of the onsite assessment, and feedback was given individually and at monthly IPC working group teleconferences. Goals of the onsite review were to assist MTF teams in conducting assessments, review practices for challenges and successes, provide on the spot education or risk mitigation, and identify common trends requiring system-wide action. RESULTS: Nine deployed MTFs participated in the onsite assessments, including four Role 3, three Role 2 capable of surgical support, and two Role 1 facilities. Seventy-eight percent of sites had assigned IPC officers although only 43% underwent required predeployment training. Hand hygiene and healthcare associated infection prevention bundles were monitored at 67% and 29% of MTFs, respectively. Several challenges including variability in practices with turnover of deployed teams were noted. Successes highlighted included individual team improvements in healthcare associated infections and mentorship of untrained personnel. CONCLUSIONS: Despite successes, ongoing challenges with optimal deployed IPC were noted. Recommendations for improvement include strengthening IPC culture, accountability, predeployment training, and stateside support for deployed IPC assets. Variability in IPC practices may occur from rotation to rotation, and regular reassessment is required to ensure that successes are sustained through times of turnover.

Characterization of sulfated polysaccharide activity against virulent Plasmodium falciparum PHISTb/RLP1 protein.

Background: The emergence of artemisinin resistance in South East Asia calls for urgent discovery of new drug compounds that have antiplasmodial activity. Unlike the classical compound screening drug discovery methods, the rational approach involving targeted drug discovery is less cumbersome and therefore key for innovation of new antiplasmodial compounds.  Plasmodium falciparum (Pf) utilizes the process of host erythrocyte remodeling using Plasmodium-helical interspersed sub-telomeric domain (PHIST) containing proteins, which are amenable drug targets. The aim of this study is to identify inhibitors of PHIST from sulfated polysaccharides as new antimalarials. Methods: 251 samples from an ongoing study of epidemiology of malaria and drug resistance sensitivity patterns in Kenya were sequenced for PHISTb/RLP1 gene using Sanger sequencing. The sequenced reads were mapped to the reference Pf3D7 protein sequence of PHISTb/RLP1 using CLC Main Workbench. Homology modeling of both reference and mutant protein structures was achieved using the LOMETs tool. The models were refined using ModRefiner for energy minimization. Ramachandran plot was generated by ProCheck to assess the conformation of amino acids in the protein model. Protein binding sites predictions were assessed using FT SITE software. We searched for prospective antimalarials from PubChem. Docking experiments were achieved using AutoDock Vina and analysis results visualized in PyMOL. Results: Sanger sequencing generated 86 complete sequences. Upon mapping of the sequences to the reference, 12 non-synonymous single nucleotide polymorphisms were considered for mutant protein structure analysis. Eleven drug compounds with antiplasmodial activity were identified. Both modelled PHISTb/RLP1 reference and mutant structures had a Ramachandran score of >90% of the amino acids in the favored region. Ten of the drug compounds interacted with amino acid residues in PHISTb and RESA domains, showing potential activity against these proteins. Conclusion: These interactions provide lead compounds for new anti-malarial molecules. Further in vivo testing is recommended.

Effects of Staphylococcus aureus Bacteriophage K on Expression of Cytokines and Activation Markers by Human Dendritic Cells In Vitro.

A potential concern with bacteriophage (phage) therapeutics is a host-versus-phage response in which the immune system may neutralize or destroy phage particles and thus impair therapeutic efficacy, or a strong inflammatory response to repeated phage exposure might endanger the patient. Current literature is discrepant with regard to the nature and magnitude of innate and adaptive immune response to phages. The purpose of this work was to study the potential effects of Staphylococcus aureus phage K on the activation of human monocyte-derived dendritic cells. Since phage K acquired from ATCC was isolated around 90 years ago, we first tested its activity against a panel of 36 diverse S. aureus clinical isolates from military patients and found that it was lytic against 30/36 (83%) of strains. Human monocyte-derived dendritic cells were used to test for an in vitro phage-specific inflammatory response. Repeated experiments demonstrated that phage K had little impact on the expression of pro- and anti-inflammatory cytokines, or on MHC-I/II and CD80/CD86 protein expression. Given that dendritic cells are potent antigen-presenting cells and messengers between the innate and the adaptive immune systems, our results suggest that phage K does not independently affect cellular immunity or has a very limited impact on it.

Complete Genome Sequence of Staphylococcus epidermidis 1457.

Staphylococcus epidermidis 1457 is a frequently utilized strain that is amenable to genetic manipulation and has been widely used for biofilm-related research. We report here the whole-genome sequence of this strain, which encodes 2,277 protein-coding genes and 81 RNAs within its 2.4-Mb genome and plasmid.

Airworthiness testing of medical maggots.

An investigation was conducted to test and certify medicinal maggots to facilitate rapid healing of traumatic and chronic wound infections in Wounded warriors being transported onboard military aircraft. Our specific aims included (1) to test the ability of medical grade larvae to withstand the rigors of U.S. Army aeromedical certification, including tolerance to change in pressure, temperature, and humidity inside ground-based chambers; (2) to evaluate the efficacy of the medical grade larvae during a high-vibration rotary-wing medical transport flight; and (3) to gain U.S. Army aeromedical certification and U.S. Air Force safe-to-fly approval and begin the steps needed to deploy/implement the use of medicinal maggots in patient care regimes for medical airlift standard operating procedures. This report outlines the ground-based and initial air-based tests performed during the study. Maggot mortality was very low during all tests, with a mortality rate of less than 1%. Maggot growth rates in wound arenas were mixed but generally depended on temperature. Overall, the results of these tests suggest that medicinal maggots can withstand the rigors of aeromedical evacuation flights in simulated flight environments and rotary- or fixed-wing aircraft.

Draft Genome Sequences of Klebsiella pneumoniae Clinical Type Strain ATCC 13883 and Three Multidrug-Resistant Clinical Isolates.

Klebsiella pneumoniae is a Gram-negative human pathogen capable of causing hospital-acquired infections with an increasing risk to human health. The total DNA from four clinically relevant strains was sequenced to >100× coverage, providing high-quality genome assemblies for K. pneumoniae strains ATCC 13883, KP4640, 101488, and 101712.