Mutation to Overproduction of Bacteriophage T4 Gene Products

R9 was isolated as one of several mutations that enhanced the growth of a leaky amber (am) mutant of bacteriophage T4 gene 62 (product required for phage DNA synthesis) under conditions of partial suppression by ribosomal ambiguity. R9 also enhanced the growth of leaky am mutants of some, but not all, other T4 “early” gene functions. R9 mapped between mutations in genes 43 and 62. By using assays involving polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate, we observed the following. (i) R9 resulted in an overproduction of many T4 “early” proteins in infected cells. The most pronounced effects of R9 were observed when phage DNA synthesis and/or the functions of maturation genes 55 and 33 were not expressed. (ii) In rifampintreated infected cells, the capacity to synthesize T4 “early” proteins decayed more slowly in the presence of the R9 mutation than in the presence of the wild-type counterpart of R9. R9 appeared to have no effect on the rates of RNA synthesis either during early or late times after infection. The results suggest that the R9 mutation leads to increased functional stability of T4 “early” messengers.     

Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and “Sarcina lutea” did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and “S. lutea” were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.     

Exercise‐induced α‐ketoglutaric acid stimulates muscle hypertrophy and fat loss through OXGR1‐dependent adrenal activation

The authors approached the journal to correct a mistake in the data presented in Appendix␣Fig S3D. The authors state that the mouse images in Appendix␣Fig S3D mistakenly displayed images from Fig 2F and Appendix␣Fig S1F. The images in Appendix␣Fig S3D are herewith corrected. The authors state that this change does not affect the conclusions or the statistics. The source data for these panels have been added to the original publication.The authors note that the following sentence needs to be corrected from: Appendix Figure S3D. Original. Appendix Figure S3D. Corrected. “Interestingly, several well‐established accumulation signatures of succinate, malate, hypoxanthine, and xanthine induced by endurance exercise (Lewis et␣al, 2010) were found to be decreased by endurance exercise (Figs 1D and EV1A–D)”.to“Interestingly, several well‐established accumulation signatures of succinate, malate, hypoxanthine, and xanthine induced by endurance exercise (Lewis et␣al, 2010) were found to be decreased by resistance exercise (Figs 1D and EV1A–D)”.Further, the authors requested to amend the legend of Appendix␣Fig S3R to indicate that the same sample for the iWAT group, “WT+2%AKG” treatment, is shown in Fig 3P. The corrected legend reads: “(R‐S). Representative images (R) and quantification (S) of p‐HSL DAB staining from male OXGR1OE^AG mice treated with AKG for 12 weeks (n = 6 per group). The same sample is shown as in Fig 3P ”.The authors regret these errors and any confusion they may have caused. All authors approve of this correction.     

The Diversity of Phaseolus-Nodulating Rhizobial Populations Is Altered by Liming of Acid Soils Planted with Phaseolus vulgaris L. in Brazil

PCR-mediated restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA internally transcribed spacer (ITS) region and the 16S rRNA gene indicated that the rhizobial populations isolated from common bean (Phaseolus vulgaris L.) nodules in the unlimed soil from a series of five lime rates applied 6 years previously to plots of an acidic oxisol had less diversity than those from plots with higher rates of liming. Isolates affiliated with Rhizobium tropici IIB and Rhizobium leguminosarum bv. phaseoli were predominant independent of lime application. An index of richness based on the number of ITS groups increased from 2.2 to 5.7 along the soil liming gradient, and the richness index based on “species” types determined by RFLP analysis of the 16S rRNA gene varied from 0.5 to 1.4. The Shannon index of diversity, based on the number of ITS groups, increased from 1.8 in unlimed soil to 2.8 in limed soil, and, based on RFLP analysis of the 16S rRNA gene, ranged from 0.9 to 1.4. In the limed soil, the subpopulation of R. tropici IIB pattern types contained the largest number of ITS groups. In contrast, there were more R. leguminosarum bv. phaseoli types in the unlimed soil with the lowest pH than in soils with the highest pH. The number of ITS (“strain”) groups within R. leguminosarum bv. phaseoli did not change with increased abundance of rhizobia in the soil, while with R. tropici IIB, the number of strain groups increased significantly. Some cultural and biochemical characteristics of Phaseolus-nodulating isolates were significantly related to changes in soil properties caused by liming, largely due to changes in the predominance of the rhizobial species groups.     

A Novel Rickettsia Species Detected in Vole Ticks (Ixodes angustus) from Western Canada

The genomic DNA of ixodid ticks from western Canada was tested by PCR for the presence of Rickettsia. No rickettsiae were detected in Ixodes sculptus, whereas 18% of the I. angustus and 42% of the Dermacentor andersoni organisms examined were PCR positive for Rickettsia. The rickettsiae from each tick species were characterized genetically using multiple genes. Rickettsiae within the D. andersoni organisms had sequences at four genes that matched those of R. peacockii. In contrast, the Rickettsia present within the larvae, nymphs, and adults of I. angustus had novel DNA sequences at four of the genes characterized compared to the sequences available from GenBank for all recognized species of Rickettsia and all other putative species within the genus. Phylogenetic analyses of the sequence data revealed that the rickettsiae in I. angustus do not belong to the spotted fever, transitional, or typhus groups of rickettsiae but are most closely related to “Candidatus Rickettsia kingi” and belong to a clade that also includes R. canadensis, “Candidatus Rickettsia tarasevichiae,” and “Candidatus Rickettsia monteiroi.”     

Cell-Specific Production and Antimicrobial Activity of Naphthoquinones in Roots of Lithospermum erythrorhizon

Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on “noninducing” medium, whereas induction of additional pigment production by abiotic (CuSO₄) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and β-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed “hairy-root” cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.     

Infection Density Dynamics of the Citrus Greening Bacterium “Candidatus Liberibacter asiaticus” in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants

Huanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, “Candidatus Liberibacter asiaticus,” and vectored by a phloem-sucking insect, Diaphorina citri. We investigated the infection density dynamics of “Ca. Liberibacter asiaticus” in field populations of D. citri with experiments using field-collected insects to address how “Ca. Liberibacter asiaticus” infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from “Ca. Liberibacter asiaticus”-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were “Ca. Liberibacter asiaticus” positive. The infections were systemic across head-thorax and abdomen, ranging from 10³ to 10⁷ bacteria per insect. In spring, the infection densities were low in March, at ∼10³ bacteria per insect, increasing up to 10⁶ to 10⁷ bacteria per insect in April and May, and decreasing to 10⁵ to 10⁶ bacteria per insect in late May, whereas the infection densities were constantly ∼10⁶ to 10⁷ bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with “Ca. Liberibacter asiaticus” infection densities in field D. citri populations. Inoculation experiments with citrus seedlings using field-collected “Ca. Liberibacter asiaticus”-infected insects suggested that (i) “Ca. Liberibacter asiaticus”-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼10⁶ bacteria per insect) of “Ca. Liberibacter asiaticus” density in D. citri is required for successful transmission to citrus plants, and (iii) D. citri attaining the threshold infection level transmits “Ca. Liberibacter asiaticus” to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen.     

Flow cytometry of spinach chloroplasts : determination of intactness and lectin-binding properties of the envelope and the thylakoid membranes

Intact spinach (Spinacia oleracea) chloroplasts, thylakoid membranes, and inside-out or right-side-out thylakoid vesicles have been characterized by flow cytometry with respect to forward angle light scatter, right angle light scatter, and chlorophyll fluorescence. Analysis of intact chloroplasts with respect to forward light scatter and the chlorophyll fluorescence parameter revealed the presence of truly “intact” and “disrupted” chloroplasts. The forward light scatter parameter, normally considered to reflect object size, was instead found to reflect the particle density. One essential advantage of flow cytometry is that additional parameters such as Ricinus communis agglutinin (linked to fluorescein isothiocyanate) fluorescence can be determined through logical conditions placed on bit-maps, amounting to an analytical purification procedure. In the present case, chloroplast subpopulations with fully preserved envelopes, thylakoid membrane, and inside-out or right-side-out thylakoid membranes vesicles can be distinguished. Flow cytometry is also a useful tool to address the question of availability of glycosyl moities on the membrane surfaces if one keeps in mind that organelle-to-organelle interactions could be partially mediated through a recognition process. A high specific binding of R. communis agglutinin and peanut lectin to the chloroplast envelope was detected. This showed that galactose residues were exposed and accessible to specific lectins on the chloroplast surface. No exposed glucose, fucose, or mannose residues could be detected by the appropriate lectins. Ricin binding to the intact chloroplasts caused a strong aggregation. Disruption of these aggregates by resuspension or during passage in the flow cytometer induced partial breakage of the chloroplasts. Only minor binding of R. communis agglutinin and peanut lectin to the purified thylakoid membranes was detected; the binding was found to be low for both inside-out and right-side-out vesicles of the thylakoid membranes.     

Biofilm regulation in Clostridioides difficile: Novel systems linked to hypervirulence

Clostridiodes difficile (C. difficile) was ranked an “urgent threat” by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the “hypervirulent” strain, {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, make it difficult to assign a “one size fits all” mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.     

Infection Dynamics of the Tick-Borne Pathogen “Candidatus Neoehrlichia mikurensis” and Coinfections with Borrelia afzelii in Bank Voles in Southern Sweden

The tick-borne bacterium “Candidatus Neoehrlichia mikurensis” has recently been recognized as a human pathogen. Together with Borrelia afzelii, it is one of the most common pathogens found in the tick Ixodes ricinus. Here, we compared the epidemiologies of “Ca. Neoehrlichia mikurensis” and B. afzelii by longitudinal sampling from May to September in one of their most abundant vertebrate hosts, the bank vole (Myodes glareolus), using real-time PCR for detection and quantification. The prevalences of “Ca. Neoehrlichia mikurensis” and B. afzelii were determined to be 19% (50/261) and 22% (56/261), respectively. The prevalence of “Ca. Neoehrlichia mikurensis” increased significantly during the sampling season. The clearance rate of “Ca. Neoehrlichia mikurensis” was significantly higher than that of B. afzelii. We found a high frequency of double infections; 46% of all samples infected with “Ca. Neoehrlichia mikurensis” also had a coinfection with B. afzelii. The frequency of coinfections was significantly higher than expected from the prevalence of each pathogen. The high level of coinfections can be caused by interactions between the pathogens or might reflect variation in general susceptibility among voles.     

A Mechanistic Model of Botrytis cinerea on Grapevines That Includes Weather,Vine Growth Stage,and the Main Infection Pathways

A mechanistic model for Botrytis cinerea on grapevine was developed. The model, which accounts for conidia production on various inoculum sources and for multiple infection pathways, considers two infection periods. During the first period (“inflorescences clearly visible” to “berries groat-sized”), the model calculates: i) infection severity on inflorescences and young clusters caused by conidia (SEV1). During the second period (“majority of berries touching” to “berries ripe for harvest”), the model calculates: ii) infection severity of ripening berries by conidia (SEV2); and iii) severity of berry-to-berry infection caused by mycelium (SEV3). The model was validated in 21 epidemics (vineyard × year combinations) between 2009 and 2014 in Italy and France. A discriminant function analysis (DFA) was used to: i) evaluate the ability of the model to predict mild, intermediate, and severe epidemics; and ii) assess how SEV1, SEV2, and SEV3 contribute to epidemics. The model correctly classified the severity of 17 of 21 epidemics. Results from DFA were also used to calculate the daily probabilities that an ongoing epidemic would be mild, intermediate, or severe. SEV1 was the most influential variable in discriminating between mild and intermediate epidemics, whereas SEV2 and SEV3 were relevant for discriminating between intermediate and severe epidemics. The model represents an improvement of previous B. cinerea models in viticulture and could be useful for making decisions about Botrytis bunch rot control.     

Genetic Diversity of African and Worldwide Strains of Ralstonia solanacearum as Determined by PCR-Restriction Fragment Length Polymorphism Analysis of the hrp Gene Region

The genetic diversity among a worldwide collection of 120 strains of Ralstonia solanacearum was assessed by restriction fragment length polymorphism (RFLP) analysis of amplified fragments from the hrp gene region. Five amplified fragments appeared to be specific to R. solanacearum. Fifteen different profiles were identified among the 120 bacterial strains, and a hierarchical cluster analysis distributed them into eight clusters. Each cluster included strains belonging to a single biovar, except for strains of biovars 3 and 4, which could not be separated. However, the biovar 1 strains showed rather extensive diversity since they were distributed into five clusters whereas the biovar 2 and the biovar 3 and 4 strains were gathered into one and two clusters, respectively. PCR-RFLP analysis of the hrp gene region confirmed the results of previous studies which split the species into an “Americanum” division including biovar 1 and 2 strains and an “Asiaticum” division including biovar 3 and 4 strains. However, the present study showed that most of the biovar 1 strains, originating from African countries (Reunion Island, Madagascar, Zimbabwe, and Angola) and being included in a separate cluster, belong to the “Asiaticum” rather than to the “Americanum” division. These African strains could thus have evolved separately from other biovar 1 strains originating from the Americas.     

Survival of Clostridium botulinum Spores

Radiation survival curves of spores of Clostridium botulinum strain 33A exhibited an exponential reduction which accounted for most of the population, followed by a “tail” comprising a very small residual number [7 to 0.7 spore(s) per ml] which resisted death in the range between 3.0 and 9.0 Mrad dose levels. The “tail” was not caused by protective spore substances released into the suspensions during irradiation, by the presence of accumulated radiation “inactivated” spores, or by heat shock of pre-irradiated spores. The theoretical number of spore targets which must be inactivated by irradiation was estimated both by a graphical and by a computation method to be about 80, and the D value was calculated to be 0.295 and 0.396 Mrad, respectively, in buffer and in pork pea broth.     

The Dispersal Ecology of Rhodesian Sleeping Sickness Following Its Introduction to a New Area

Tsetse-transmitted human and animal trypanosomiasis are constraints to both human and animal health in sub-Saharan Africa, and although these diseases have been known for over a century, there is little recent evidence demonstrating how the parasites circulate in natural hosts and ecosystems. The spread of Rhodesian sleeping sickness (caused by Trypanosoma brucei rhodesiense) within Uganda over the past 15 years has been linked to the movement of infected, untreated livestock (the predominant reservoir) from endemic areas. However, despite an understanding of the environmental dependencies of sleeping sickness, little research has focused on the environmental factors controlling transmission establishment or the spatially heterogeneous dispersal of disease following a new introduction. In the current study, an annually stratified case-control study of Rhodesian sleeping sickness cases from Serere District, Uganda was used to allow the temporal assessment of correlations between the spatial distribution of sleeping sickness and landscape factors. Significant relationships were detected between Rhodesian sleeping sickness and selected factors, including elevation and the proportion of land which was “seasonally flooding grassland” or “woodlands and dense savannah.” Temporal trends in these relationships were detected, illustrating the dispersal of Rhodesian sleeping sickness into more ‘suitable’ areas over time, with diminishing dependence on the point of introduction in concurrence with an increasing dependence on environmental and landscape factors. These results provide a novel insight into the ecology of Rhodesian sleeping sickness dispersal and may contribute towards the implementation of evidence-based control measures to prevent its further spread.     

Monitoring cellular redox dynamics using newly developed BRET-based redox sensor proteins

Reactive oxygen species are key factors that strongly affect the cellular redox state and regulate various physiological and cellular phenomena. To monitor changes in the redox state, we previously developed fluorescent redox sensors named Re-Q, the emissions of which are quenched under reduced conditions. However, such fluorescent probes are unsuitable for use in the cells of photosynthetic organisms because they require photoexcitation that may change intracellular conditions and induce autofluorescence, primarily in chlorophylls. In addition, the presence of various chromophore pigments may interfere with fluorescence-based measurements because of their strong absorbance. To overcome these problems, we adopted the bioluminescence resonance energy transfer (BRET) mechanism for the sensor and developed two BRET-based redox sensors by fusing cyan fluorescent protein–based or yellow fluorescent protein–based Re-Q with the luminescent protein Nluc. We named the resulting redox-sensitive BRET-based indicator probes “ROBINc” and “ROBINy.” ROBINc is pH insensitive, which is especially vital for observation in photosynthetic organisms. By using these sensors, we successfully observed dynamic redox changes caused by an anticancer agent in HeLa cells and light/dark-dependent redox changes in the cells of photosynthetic cyanobacterium Synechocystis sp. PCC 6803. Since the newly developed sensors do not require excitation light, they should be especially useful for visualizing intracellular phenomena caused by redox changes in cells containing colored pigments.     

Non-Listening and Self Centered Leadership – Relationships to Socioeconomic Conditions and Employee Mental Health

Background

The way in which leadership is experienced in different socioeconomic strata is of interest per se, as well as how it relates to employee mental health.

Methods

Three waves of SLOSH (Swedish Longitudinal Occupational Survey of Health, a questionnaire survey on a sample of the Swedish working population) were used, 2006, 2008 and 2010 (n = 5141). The leadership variables were: “Non-listening leadership” (one question: “Does your manager listen to you?” - four response categories), “Self centered leadership” (sum of three five-graded questions – “non-participating”, “asocial” and “loner”). The socioeconomic factors were education and income. Emotional exhaustion and depressive symptoms were used as indicators of mental health.

Results

Non-listening leadership was associated with low income and low education whereas self-centered leadership showed a weaker relationship with education and no association at all with income. Both leadership variables were significantly associated with emotional exhaustion and depressive symptoms. “Self centered” as well as “non-listening” leadership in 2006 significantly predicted employee depressive symptoms in 2008 after adjustment for demographic variables. These predictions became non-significant when adjustment was made for job conditions (demands and decision latitude) in the “non-listening” leadership analyses, whereas predictions of depressive symptoms remained significant after these adjustments in the “self-centered leadership” analyses.

Conclusions

Our results show that the leadership variables are associated with socioeconomic status and employee mental health. “Non-listening” scores were more sensitive to societal change and more strongly related to socioeconomic factors and job conditions than “self-centered” scores.     

Higher Landing Accuracy in Expert Pilots is Associated with Lower Activity in the Caudate Nucleus

The most common lethal accidents in General Aviation are caused by improperly executed landing approaches in which a pilot descends below the minimum safe altitude without proper visual references. To understand how expertise might reduce such erroneous decision-making, we examined relevant neural processes in pilots performing a simulated landing approach inside a functional MRI scanner. Pilots (aged 20–66) were asked to “fly” a series of simulated “cockpit view” instrument landing scenarios in an MRI scanner. The scenarios were either high risk (heavy fog–legally unsafe to land) or low risk (medium fog–legally safe to land). Pilots with one of two levels of expertise participated: Moderate Expertise (Instrument Flight Rules pilots, n = 8) or High Expertise (Certified Instrument Flight Instructors or Air-Transport Pilots, n = 12). High Expertise pilots were more accurate than Moderate Expertise pilots in making a “land” versus “do not land” decision (CFII: d′ = 3.62±2.52; IFR: d′ = 0.98±1.04; p<.01). Brain activity in bilateral caudate nucleus was examined for main effects of expertise during a “land” versus “do not land” decision with the no-decision control condition modeled as baseline. In making landing decisions, High Expertise pilots showed lower activation in the bilateral caudate nucleus (0.97±0.80) compared to Moderate Expertise pilots (1.91±1.16) (p<.05). These findings provide evidence for increased “neural efficiency” in High Expertise pilots relative to Moderate Expertise pilots. During an instrument approach the pilot is engaged in detailed examination of flight instruments while monitoring certain visual references for making landing decisions. The caudate nucleus regulates saccade eye control of gaze, the brain area where the “expertise” effect was observed. These data provide evidence that performing “real world” aviation tasks in an fMRI provide objective data regarding the relative expertise of pilots and brain regions involved in it.     

Drug Resistance in Natural Isolates of Leishmania donovani s.l. Promastigotes Is Dependent of Pgp170 Expression

Resistance of pathogens to drugs is a growing concern regarding many diseases. Parasites like Leishmania, Plasmodium and Entamoeba histolytica; and neoplastic cells, present the multidrug-resistant phenotype rendering chemotherapy ineffective. The acquired resistance of Leishmania to antimony has generated intense research on the mechanisms involved but the question has not yet been resolved. To test the hypothesis that drug efflux in Leishmania, as measured by flow cytometry using the fluorescent dye Rhodamine-123, is largely dependent on the number of efflux pumps an isolate can express, the amount of Pgp 170 molecules was assessed in ten field isolates (5 “resistant” and 5 “susceptible”) using: Western Blotting, Confocal and Transmission Electron Microscopy, and proteomics. Their survival after exposure to three antileishmanial drugs, in vitro, was evaluated and clinical data were compared to the in vitro results. All isolates were resistant to Glucantime but susceptible to Miltefosine, whilst Amphotericin B was more effective on the “susceptible” isolates. The MDR gene, expressing the transmembrane efflux pump Pgp 170, appears to play a key role in the phenomenon of drug resistance. When “susceptible” versus “resistant” parasites were compared, it was shown that the higher the number of Pgp 170 molecules the higher the Rhodamine-123 efflux from the parasite body and, when exposed to the drug, the number of efflux pumps increased. However, the rate of this increase was not linear and it is possible that there is a maximum number of Pgp 170 molecules an isolate can express. Nevertheless, the phenomenon is a complex one and other factors and proteins are involved in which the HSP-70 group proteins, detected in the “resistant” isolates, may play a significant role.