Engineering and functional analysis of yeast with a monotypic 40S ribosome subunit.

Emerging evidence reveals that ribosomes are not monolithic but dynamic machines with heterogeneous protein compositions that can reshape ribosomal translational abilities and cellular adaptation to environmental changes. Duplications of ribosomal protein (RP) genes are ubiquitous among organisms and are believed to affect cell function through paralog-specific regulation (e.g., by generating heterogeneous ribosomes) and/or gene dose amplification. However, direct evaluations of their impacts on cell function remain elusive due to the highly heterogeneous cellular RP pool. Here, we engineered a yeast with homogeneous 40S RP paralog compositions, designated homo-40S, by deleting the entire set of alternative duplicated genes encoding yeast 40S RP paralogs. Homo-40S displayed mild growth defects along with high sensitivity to the translation inhibitor paromomycin and a significantly increased stop codon readthrough. Moreover, doubling of the remaining RP paralogous genes in homo-40S rescued these phenotypes markedly, although not fully, compared to the wild-type phenotype, indicating that the dose of 40S RP genes together with the heterogeneity of the contents was vital for maintaining normal translational functionalities and growth robustness. Additional experiments revealed that homo-40S improved paromomycin tolerance via acquisition of bypass mutations or evolved to be diploid to generate fast-growing derivatives, highlighting the mutational robustness of engineered yeast to accommodate environmental and genetic changes. In summary, our work demonstrated that duplicated RP paralogs impart robustness and phenotypic plasticity through both gene dose amplification and paralog-specific regulation, paving the way for the direct study of ribosome biology through monotypic ribosomes with a homogeneous composition of specific RP paralogs.

Disease-specific differences in pharmacokinetics of paromomycin and miltefosine between post-kala-azar dermal leishmaniasis and visceral leishmaniasis patients in eastern Africa.

Treatment regimens for post-kala-azar dermal leishmaniasis (PKDL) are usually extrapolated from those for visceral leishmaniasis (VL), but drug pharmacokinetics (PK) can differ due to disease-specific variations in absorption, distribution, and elimination. This study characterized PK differences in paromomycin and miltefosine between 109 PKDL and 264 VL patients from eastern Africa. VL patients showed 0.55-fold (95%CI: 0.41-0.74) lower capacity for paromomycin saturable reabsorption in renal tubules, and required a 1.44-fold (1.23-1.71) adjustment when relating renal clearance to creatinine-based eGFR. Miltefosine bioavailability in VL patients was lowered by 69% (62-76) at treatment start. Comparing PKDL to VL patients on the same regimen, paromomycin plasma exposures were 0.74-0.87-fold, while miltefosine exposure until the end of treatment day was 1.4-fold. These pronounced PK differences between PKDL and VL patients in eastern Africa highlight the challenges of directly extrapolating dosing regimens from one leishmaniasis presentation to another.

Positive clinical experience with paromomycin sulfate in treatment of Balantioides coli (=Balantidium coli) natural infection in zoo-kept Mandrill monkeys (Mandrillus sphinx) and Western lowland gorillas (Gorilla gorilla gorilla).

Balantioides coli (=Balantidium coli), a large ciliated protozoan, is reported in multiple free-ranging and captive primate species, often in association with a clinical presentation that requires medical intervention. This report describes the clinical effectiveness of paromomycin sulfate against B.coli in zoo-kept mandrill monkeys (Mandrillus sphinx, at orally doses of 8-31 mg/kg, once daily (SID) for 7 days) and gorillas (Gorilla gorilla gorilla, at orally doses of 1.4-3.1 mg/kg, SID for 5 days).

Paromomycin and Miltefosine Combination as an Alternative to Treat Patients With Visceral Leishmaniasis in Eastern Africa: A Randomized, Controlled, Multicountry Trial.

BACKGROUND: This study aimed to determine whether paromomycin plus miltefosine (PM/MF) is noninferior to sodium stibogluconate plus paromomycin (SSG/PM) for treatment of primary visceral leishmaniasis in eastern Africa. METHODS: An open-label, phase 3, randomized, controlled trial was conducted in adult and pediatric patients at 7 sites in eastern Africa. Patients were randomly assigned to either 20 mg/kg paromomycin plus allometric dose of miltefosine (14 days), or 20 mg/kg sodium stibogluconate plus 15 mg/kg paromomycin (17 days). The primary endpoint was definitive cure after 6 months. RESULTS: Of 439 randomized patients, 424 completed the trial. Definitive cure at 6 months was 91.2% (155 of 170) and 91.8% (156 of 170) in the PM/MF and SSG/PM arms in primary efficacy modified intention-to-treat analysis (difference, 0.6%; 97.5% confidence interval [CI], -6.2 to 7.4), narrowly missing the noninferiority margin of 7%. In the per-protocol analysis, efficacy was 92% (149 of 162) and 91.7% (155 of 169) in the PM/MF and SSG/PM arms (difference, -0.3%; 97.5% CI, -7.0 to 6.5), demonstrating noninferiority. Treatments were well tolerated. Four of 18 serious adverse events were study drug-related, and 1 death was SSG-related. Allometric dosing ensured similar MF exposure in children (<12 years) and adults. CONCLUSIONS: PM/MF and SSG/PM efficacies were similar, and adverse drug reactions were as expected given the drugs safety profiles. With 1 less injection each day, reduced treatment duration, and no risk of SSG-associated life-threatening cardiotoxicity, PM/MF is a more patient-friendly alternative for children and adults with primary visceral leishmaniasis in eastern Africa. CLINICAL TRIALS REGISTRATION: NCT03129646.

Highly sensitive liquid chromatographic method combined with online ion suppression to remove interfering anions and mass spectrometry for impurity profiling of paromomycin sulfate.

A previously developed high-performance liquid chromatography method combined with pulsed amperometric detection allowed to separate many impurities of paromomycin. However, due to the presence of ion pairing agents and sodium hydroxide in the mobile phase, direct coupling to mass spectrometry for the identification of the chemical structures of the impurities was not an option. Indeed, ion suppression was encountered by trifluoroacetic acid and pentafluoroproponic acid in the mobile phase. A cation self-regenerating suppressor, which was originally designed for increasing analyte conductivity of ammonia and amines analysis in ion chromatography, was coupled between the liquid chromatography and ion trap-time of flight-mass spectrometry and almost all trifluoroacetic acid and pentafluoroproponic acid in the mobile phase was removed. The limit of detection of paromomycin in this integrated system improved significantly to 20 ng/ml (0.4 ng). The chemical structures of 19 impurities were elucidated and seven impurities were reported for the first time.

Targeted modifications of neomycin and paromomycin: Towards resistance-free antibiotics?

Despite their clinical importance, saving numerous human lifes, over- and mis-uses of antibiotics have created a strong selective pressure on bacteria, which induces the emergence of (multi)resistant strains. Antibioresistance is becoming so pregnant that since 2017, WHO lists bacteria threatening most human health (AWaRe, ESKAPE lists), and those for which new antibiotics are urgently needed. Since the century turn, this context is leading to a burst in the chemical synthesis of new antibiotics, mostly derived from natural antibiotics. Among them, aminoglycosides, and especially the neomycin family, exhibit broad spectrum of activity and remain clinically useful drugs. Therefore, numerous endeavours have been undertaken to modify aminoglycosides with the aim of overcoming bacterial resistances. After having replaced antibiotic discovery into an historical perspective, briefly surveyed the aminoglycoside mode of action and the associated resistance mechanisms, this review emphasized the chemical syntheses performed on the neomycin family and the corresponding structure activity relationships in order to reveal the really efficient modifications able to convert neomycin and its analogues into future drugs. This review would help researchers to strategically design novel aminoglycoside derivatives for the development of clinically viable drug candidates.

Genome-wide analysis reveals allelic variation and chromosome copy number variation in paromomycin-resistant Leishmania donovani.

In the absence of adequate diagnosis and treatment, leishmaniasis remains a major public health concern on a global scale. Drug resistance remains a key obstacle in controlling and eliminating visceral leishmaniasis. The therapeutic gap due to lack of target-specific medicine and vaccine can be minimized by obtaining parasite's genomic information. This study compared whole-genome sequence of paromomycin-resistant parasite (K133PMM) developed through in vitro adaptation and selection with sensitive Leishmania clinical isolate (K133WT). We found a large number of upstream and intergenic gene variations in K133PMM. There were 259 single nucleotide polymorphisms (SNPs), 187 insertion-deletion (InDels), and 546 copy number variations (CNVs) identified. Most of the genomic variations were found in the gene's upstream and non-coding regions. Ploidy estimation revealed chromosome 5 in tetrasomy and 6, 9, and 12 in trisomy, uniquely in K133PMM. These contain the genes for protein degradation, parasite motility, autophagy, cell cycle maintenance, and drug efflux membrane transporters. Furthermore, we also observed reduction in ploidy of chromosomes 15, 20, and 23, in the resistant parasite containing mostly the genes for hypothetical proteins and membrane transporters. We chronicled correlated genomic conversion and aneuploidy in parasites and hypothesize that this led to rapid evolutionary changes in response to drug induced pressure, which causes them to become resistant.

Isolation, typing, and drug susceptibility of Leishmania (Leishmania) infantum isolates from dogs of the municipality of Embu das Artes, an endemic region for canine leishmaniasis in Brazil.

The parasitic protozoa Leishmania (Leishmania) infantum is the etiological agent of human visceral leishmaniasis and canine leishmaniasis in South America, where Brazil is the most affected country. This zoonotic disease is transmitted by the bite of an infected phlebotomine sand fly and dogs constitute the main domestic reservoir of the parasite. In this study, we screened 2348 dogs of the municipality of Embu das Artes, Brazil, for antibodies against the parasite. Prevalence for canine leishmaniasis seropositivity was 2.81%, as assessed using a Dual-Path Platform rapid test for canine leishmaniasis. Twenty-five seropositive dogs were euthanized for parasite isolation and 14 isolates were successful obtained. Nucleotide sequencing of the internal transcribed spacer confirmed the isolates to be L. (L.) infantum, and very low sequence variability was observed among them. The in vitro susceptibility to miltefosine and paromomycin was assessed and moderate variation in paromomycin susceptibility was found among the isolates in the promastigote and intracellular amastigote stages. On the other hand, in vitro susceptibility to miltefosine of these isolates was homogenous, particularly in the amastigote stage (EC50 values from 0.69 to 2.07 µM). In addition, the miltefosine sensitivity locus was deleted in all the isolates, which does not corroborate the hypothesis that the absence of this locus is correlated with a low in vitro susceptibility. Our findings confirm that the municipality of Embu das Artes is endemic for canine leishmaniasis and that isolates from this region are susceptible to paromomycin and miltefosine, indicating the potential of these drugs to be clinically evaluated in the treatment of human visceral leishmaniasis in Brazil.

Production and statistical optimization of Paromomycin by Streptomyces rimosus NRRL 2455 in solid state fermentation.

BACKGROUND: Paromomycin is a 2-deoxystreptamine aminocyclitol aminoglycoside antibiotic with broad spectrum activity against Gram-negative, Gram-positive bacteria and many protozoa. This study introduces a strategy for paromomycin production through solid-state fermentation using Streptomyces rimosus subsp. paromomycinus NRRL 2455. Solid state fermentation has gained enormous attention in the development of several products because of their numerous advantages over submerged liquid fermentation. After selecting the best solid substrate, a time course study of paromomycin production was carried out followed by optimization of environmental conditions using response surface methodology. Paromomycin yields obtained using this technique were also compared to those obtained using submerged liquid fermentation. RESULTS: Upon screening of 6 different substrates, maximum paromomycin concentration (0.51 mg/g initial dry solids) was obtained with the cost-effective agro-industrial byproduct, corn bran, impregnated with aminoglycoside production media. Optimization of environmental conditions using D-optimal design yielded a 4.3-fold enhancement in paromomycin concentration reaching 2.21 mg/g initial dry solids at a pH of 8.5, inoculum size of 5% v/w and a temperature of 30 °C. CONCLUSION: Compared to submerged liquid fermentation, solid state fermentation resulted in comparable paromomycin concentrations, cost reduction of raw materials, less energy consumption and waste water discharge, which have major implications in industrial fermentation. Therefore, solid state fermentation is a promising alternative to submerged liquid fermentation for paromomycin production. To the best of our knowledge, this is the first report on the optimized paromomycin production through solid state fermentation process.

Mechanical plus oral bowel preparation with paromomycin and metronidazole reduces infectious complications in elective colorectal surgery: a matched case-control study.

PURPOSE: Infectious complications are as high as 30% in elective colorectal surgery. In recent years, several studies have discussed the topic of preoperative bowel decontamination prior to colorectal surgery in order to reduce postoperative infectious complications and have found significant effects of oral antibiotic administration with a large variety of drugs used. No study has evaluated the combination of oral paromomycin and metronidazole in this context. METHODS: We performed a prospective single-center study with a matched-pair retrospective cohort to evaluate postoperative infectious complications (superficial site infections, organ space abscess, anastomotic leakage) in elective colorectal surgery. PATIENTS: A total of 120 patients were available for study inclusion; 101 gave informed consent and were included. A total of 92 patients were matched and subsequently analyzed. We could show a reduction in overall infectious complications in the intervention group (15.2% vs 30.8%, p = 0.018; odds ratio 0.333, 95% CI 0.142-0.784) as well as a reduction in superficial surgical site infections (8.7 vs 19.6%, p = 0.041, OR 0.333, 95% CI 0.121-0.917). The frequency of the other infectious complications such as intraabdominal abscesses and anastomotic leakage showed a tendency towards decreased frequencies in the intervention group (OR 0.714, 95% CI 0.235-2.169 and OR 0.571; 95% CI 0.167-1.952, respectively). Finally, the oral antibiotic administration led to an almost significantly reduced length of stay (12.24 days vs 15.25 days; p = 0.057). CONCLUSIONS: Oral paromomycin and metronidazole with intravenous ertapenem effectively reduce infectious complications in elective colorectal surgery. TRIAL REGISTRATION: The study was registered at Clinicaltrials.gov (NCT03759886) December 17, 2018.

Antileishmanial efficacy and tolerability of combined treatment with non-ionic surfactant vesicle formulations of sodium stibogluconate and paromomycin in dogs.

Infection with Leishmania infantum causes the disease visceral leishmaniasis (VL), which is a serious clinical and veterinary problem. The drugs used to treat canine leishmaniasis (CanL) do not cause complete parasite clearance; they can be toxic, and emerging drug resistance in parasite populations limits their clinical utility. Therefore, in this study we have evaluated the toxicity and efficacy of joint treatment with a 1:1 mixture of sodium stibogluconate-NIV (SSG-NIV, 10 mg Sbv/day) and paromomycin-NIV (PMM-NIV, 10 mg PMM/kg/day), given intravenously daily for seven days from day 270 post-infection, to nine-month-old female beagle dogs (n = 6) experimentally infected with Leishmania infantum. Treatment significantly improved the clinical symptoms of VL infection in all the treated dogs, reduced parasite burdens in lymph nodes and bone marrow, and all symptomatic treated dogs, were asymptomatic at 90 days post-treatment. Treatment was associated with a progressive and significant decrease in specific IgG anti-Leishmania antibodies using parasite soluble antigen (p < 0.01) or rK39 (p < 0.01) as the target antigen. In addition, all dogs were classified as parasite negative based on Leishmania nested PCR and quantitative real time PCR tests and as well as an inability to culture of promastigote parasites from lymph nodes and bone marrow tissue samples taken at day 90 post-treatment. However, treatment did not cure the dogs as parasites were detected at 10 months post-treatment, indicating that a different dosing regimen is required to cause long term cure or prevent relapse.

Development of amoebic liver abscess in early pregnancy years after initial amoebic exposure: a case report.

BACKGROUND: Infection with Entamoeba histolytica and associated complications are relatively rare in developed countries. The overall low prevalence in the Western world as well as the possibly prolonged latency period between infection with the causing pathogen and onset of clinical symptoms may delay diagnosis of and adequate treatment for amoebiasis. Amoebic liver abscess (ALA) is the most common extraintestinal manifestation of invasive amoebiasis. Pregnancy has been described as a risk factor for development of invasive amoebiasis and management of these patients is especially complex. CASE PRESENTATION: A 30-year-old Caucasian woman in early pregnancy presented to our emergency department with abdominal pain alongside elevated inflammatory markers and liver function tests. Travel history revealed multiple journeys to tropic and subtropic regions during the past decade and a prolonged episode of intermittently bloody diarrhea during a five month stay in Indonesia seven years prior to admission. Sonographic and magnetic resonance imaging revealed a 5 × 4 cm hepatic abscess. After ultrasound-guided transcutaneous liver drainage, both abscess fluids and blood cultures showed neither bacterial growth nor microscopic signs of parasitic disease. Serological testing confirmed an infection with Entamoeba histolytica, which was treated with metronidazole, followed by eradication therapy with paromomycin. Subsequent clinical, laboratory and imaging follow-up exams showed regression of the ALA. In addition, the pregnancy completed without complications and a healthy baby boy was born 7 months after termination of treatment. CONCLUSIONS: This case of invasive amoebiasis in early pregnancy outside of endemic regions and several years after exposure demonstrates the importance of broad differential diagnostics in the context of liver abscesses. The complex interdisciplinary decisions regarding the choice of imaging techniques as well as interventional and antibiotic therapy in the context of pregnancy are discussed. Furthermore, we present possible explanations for pregnancy as a risk factor for an invasive course of amoebiasis.

Spotlight on Histomonosis (blackhead disease): a re-emerging disease in turkeys and chickens.

Histomonosis, or blackhead disease, is a well-known disease in turkeys that can cause high mortality, but outbreaks with lower losses are also observed. The disease is less fatal in chickens but is economically important due to reduced performance and its co-appearance with colibacillosis. The lack of specific prophylactic and therapeutic interventions has led to a re-emergence of the disease in recent years, mainly in turkeys, free-range layers and chicken parent stock.

A randomized, blinded, controlled clinical trial comparing the efficacy of aminosidine (paromomycin)-allopurinol combination with the efficacy of meglumine antimoniate-allopurinol combination for the treatment of canine leishmaniosis due to Leishmania infantum.

The aim of this 6-month, randomized, blinded, controlled clinical trial was to compare the efficacy and safety of aminosidine-allopurinol combination with that of meglumine antimoniate-allopurinol combination for the treatment of leishmaniosis in dogs without stage III or IV chronic kidney disease. Forty client-owned dogs were randomly assigned to group A [n = 20; aminosidine (15 mg/kg, subcutaneously, once daily, for 28 days) and allopurinol (10 mg/kg, per os, twice daily, for 6 months)] or group B [(n = 20; meglumine antimoniate (100 mg/kg SC, once daily, for 28 days) and allopurinol (10 mg/kg, per os, twice daily, for 6 months)]. Clinical and clinicopathological evaluations, parasitic load measurement (lymph node and bone marrow microscopy, bone marrow real-time PCR), specific serology and leishmanin skin test (LST) were performed at baseline (time 1) and after 14 (time 2), 28 (time 3), 60 (time 4) and 180 (time 5) days. Both treatments were safe and resulted in significant clinical and clinicopathological improvement, reduction of parasitic load and of indirect immunofluorescence antibody test (IFAT) titer and induction of positive LST. There was no significant difference between groups with regards to the primary outcome measures of the trial that included the proportion of dogs that presented severe treatment-related side effects, were cured and were parasitologically negative at time 5. However, some (proportion of dogs that presented no clinical signs, no hyperglobulinemia and negative serology at time 5) secondary outcome measures showed significant differences in favor of the meglumine antimoniate-allopurinol treatment arm. Treatment-related death occurred in one dog in each group, while injection site reactions appeared at a similar frequency in both groups. Due to the differences in some secondary outcome measures in association with the low power of this trial, it cannot be definitively concluded that the two treatments are equally effective. Therefore, the aminisodine-allopurinol combination cannot be proposed as a first-line treatment of CanL but rather as a second-line treatment that may be particularly useful to avoid repeated administration of meglumine antimoniate and in countries where the latter is not available or registered.

Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae.

Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3'-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter.

Topical 15% Paromomycin-Aquaphilic for Bolivian Leishmania braziliensis Cutaneous Leishmaniasis: A Randomized, Placebo-controlled Trial.

BACKGROUND: Cutaneous leishmaniasis (CL) presents as 1 or more skin lesions, which makes local therapy inherently attractive compared to systemic therapy that exposes the whole body to a drug. For 30 years, 15% paromomycin topical formulations have been in clinical experimentation. Recently, 15% paromomycin in Aquaphilic, a complex base to facilitate adsorption into the lesion, was found superior to aquaphilic vehicle for Old World Leishmania major disease. METHODS: We performed a randomized trial of 15% paromomycin in Aquaphilic (40 patients) vs Aquaphilic vehicle (20 patients) vs a positive control (intralesional pentamidine; 20 patients) against L. braziliensis CL in Bolivia. RESULTS: Cure rates after 6 months of follow-up were 31 of 40 (77.5%, 95% confidence interval [CI] 62.5-88%) for paromomycin-Aquaphilic, 2 of 20 (10%, 95% CI 3-30%) for Aquaphilic vehicle (P < .0001 vs paromomycin-Aquaphilic), and 14 of 20 (70%, 95% CI 48-85.5%) for intralesional pentamidine. Both paromomycin-Aquaphilic and the Aquaphilic vehicle were very well tolerated, with only grade 1 adverse reactions in 5-10% of patients. CONCLUSIONS: Against L. braziliensis CL, a prevalent, aggressive form of New World CL, 15% paromomycin-aquaphilic was vastly superior to a negative vehicle control and was comparable in efficacy to a positive control. This study enlarges the potential use of 15% paromomycin-Aquaphilic from one form of Old World CL to CL more generally. CLINICAL TRIALS REGISTRATION: NCT03096457.

Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains.

Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMMr) was induced in three Nepalese clinical strains of Leishmania donovani with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites, even though a single cloned population was used at the start of selection. PMM 50% inhibitory concentration (IC50) values for PMMr parasites varied between 104 and 481 µM at the promastigote stage and 32 and 195 µM at the intracellular amastigote stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote stage but not the promastigote stage (P < 0.05). This effect was most marked in the Sb-resistant (Sbr) PMMr clone, in which PMM resistance was associated with a significant upregulation of glutathione compared to that in its wild type (P < 0.05), although there was no change in the regulation of trypanothione (detected in its oxidized form). Interestingly, PMMr strains showed an increase in either the keto acid derivative of isoleucine (Sb intermediate PMMr) or the 2-hydroxy acids derived from arginine and tyrosine (Sb susceptible PMMr and Sbr PMMr). These results are consistent with the recent finding that the upregulation of the branched-chain amino acid aminotransferase and d-lactate dehydrogenase is linked to PMMr In addition, we found that PMMr is associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow the rapid selection of genetic changes that confer a survival advantage.

Chemogenomic Profiling of Antileishmanial Efficacy and Resistance in the Related Kinetoplastid Parasite Trypanosoma brucei.

The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode of action and potential routes to resistance is limited. Forward genetic approaches have revolutionized our understanding of drug mode of action in the related kinetoplastid parasite Trypanosoma brucei Therefore, we screened our genome-scale T. brucei RNA interference (RNAi) library against the current antileishmanial drugs sodium stibogluconate (antimonial), paromomycin, miltefosine, and amphotericin B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin-aminoglycoside efficacy. The vesicle-associated membrane protein TbVAMP7B and a flippase contribute to amphotericin B and miltefosine action and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative ß-subunit/CDC50 cofactor, and additional membrane-associated hits, affect amphotericin B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of antileishmanial drug action and potential resistance mechanisms and should facilitate the development of improved therapies as well as surveillance for drug-resistant parasites.

Paromomycin production from Streptomyces rimosus NRRL 2455: statistical optimization and new synergistic antibiotic combinations against multidrug resistant pathogens.

BACKGROUND: Response surface methodology (RSM) employing Box-Behnken design was used to optimize the environmental factors for the production of paromomycin, a 2 deoxystreptamine aminocyclitol aminoglycoside antibiotic, (2DOS-ACAGA) from Streptomyces (S.) rimosus NRRL 2455. Emergence of bacterial resistance caught our attention to consider the combination of antimicrobial agents. The effect of paromomycin combination with other antimicrobial agents was tested on some multiple drug resistant isolates. To the best of our knowledge, this is the first report on optimization of paromomycin production from S. rimosus NRRL 2455. A Quadratic model and response surface method were used by choosing three model factors; pH, incubation time and inoculum size. A total of 17 experiments were done and the response of each experiment was recorded. Concerning the effect of combining paromomycin with different antimicrobial agents, it was tested using the checkerboard assay against six multidrug resistant (MDR) pathogens including; Pseudomonas (P.) aeruginosa (2 isolates), Klebsiella (K.) pneumoniae, Escherichia (E.) coli, methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA). Paromomycin was tested in combination with ceftriaxone, ciprofloxacin, ampicillin/sulbactam, azithromycin, clindamycin and doxycycline. RESULTS: The optimum conditions for paromomycin production were a pH of 6, an incubation time of 8.5 days and an inoculum size of 5.5% v/v using the optimized media (soybean meal 30 g/L, NH4CL 4 g/L, CaCO3 5 g/L and glycerol 40 ml/L), 28 °C incubation temperature, and 200 rpm agitation rate that resulted in 14 fold increase in paromomycin production as compared to preliminary fermentation level using the basal medium. The tested antibiotic combinations showed either synergistic effect on paromomycin activity on most of the tested MDR pathogens (45.83%), additive effect in 41.67% or indifferent effect in 12.5%. CONCLUSION: RSM using multifactorial design was a helpful and a reliable method for paromomycin production. Paromomycin combination with ceftriaxone, ciprofloxacin, ampicillin/sulbactam, azithromycin, clindamycin or doxycycline showed mostly synergistic effect on certain selected clinically important MDR pathogens.

Evaluation of paromomycin sulphate permeation using ex vivo human skin model.

Ex vivo evaluation of drug release and skin permeation from topical formulations of antileishmanial drug paromomycin sulphate was carried out using intact full thickness human skin. Potency-based microbiological assay was used for the analysis of paromomycin concentrations. A total percentage drug recovery of 86 ± 26% was obtained. Incubation periods of 1 and 3 h resulted in percentage drug permeation into deep skin layers ranging from 1.3 ± 0.04% to 5.3 ± 2.0% with paraffin-based ointment and from 1.6 ± 0.8% to 3.9 ± 1% with microemulsion-based emulgel. Although a small percentage, this is still significantly higher than those previously reported using animal skin models.