Presence Of Efflux Pumps Research Articles

Molecular mechanisms of biofilm resistance against antibiotics

Biofilms are immobile communities of microbes attached to biotic an abiotic surfaces and are embedded inside a self-produced cement-like extracellular polymeric substances. The resistance of biofilms against commonly used drugs has been implicated in the pathogenesis of various bacterial infections under medical and veterinary settings which normally cannot be eradicated by antibiotics. Biofilms are characterized by the ability to evade not only the antibiotic effects but also the host immune system clearance. Currently the most worrisome aspect of global human health is the rise and spread of antimicrobial resistance in bacterial pathogens and this crisis got deepened by the emergence of antimicrobial resistance of bacterial biofilms. Different antibiotic resistance mechanisms, processes by which a target pathogen curtails the interaction between an antimicrobial agent and its intended target molecules, adopted by biofilms have been discussed in this review. Different antibiotic resistance mechanisms are employed by the biofilms depending on the species of the bacteria, growth conditions, and the antibiotic involved. Commonly, the role of biofilm matrix polysaccharides, antibiotic-modifying or degrading enzymes, extracellular DNA, hypoxic conditions, presence of efflux pumps, quorum sensing, horizontal gene transfer, mutation frequency, etc. have been implicated in antibiotic resistance of biofilms. This review also discusses different approaches of overcoming biofilm infections or biofilm resistance. However, it is pertinent to mention that since no new class of antibiotics have been approved in last four decades there is the need of greater understanding of biofilm-associated antibiotic resistance to effectively utilise the therapeutic value of the existing antibiotics. Although a number of anti-biofilm strategies have been put forward as discussed in this review, they are still in nascent stage and need to undergo clinical trials to reach the commercial market.

RESISTANCE MECHANISMS OF MYCOBACTERIUM TUBERCULOSIS IN THE TUBERCULOSIS PATHOGENICITY IN HUMANS: AN OVERVIEW

Tuberculosis (TB) is a chronic and serious infectious disease caused by Mycobacterium tuberculosis that mainly affects the lungs, but it is also capable of infecting other organs/tissues in the body, which triggers extra-pulmonary TB. In recent years, there has been an increase in the number of re-emerging TB cases due to resistance of M. tuberculosis to the antibiotics used to treat this disease. On top of that, there is the emergence of multidrug-resistant M. tuberculosis for which many of the antibiotics previously used have no effect. For these reasons, it was aimed to investigate the resistance mechanisms of M. tuberculosis in the TB pathogenicity in humans, considering this as a factor associated with re-emergence cases of this disease. Thus, it was carried out an integrative literature review based on articles selected in the SciElo, PubMed and Science Direct databases by using terminology in Portuguese and in English. Studies highlighted that the resistance mechanisms of M. tuberculosis consist of the emergence of mutations in target-genes naturally selected over time, the presence of efflux pumps and some lipid substances in the bacterial cell wall that provide impermeability to the antibiotics with cytoplasmic activity. Furthermore, the inappropriate use of antibiotics during the antibiotic-therapy period causes the emergence of other types of resistance. Therefore, special attention must be given to the correct use of antibiotics during the TB treatment, as well as the development of new effective drugs against multidrug-resistant bacteria strains.

Detection of proper Type 3 Secretion System Translocon Assembly using a split Luciferase Assay

Type 3 secretion system (T3SS) is used by many Gram‐negative bacteria to colonize the host and inject effector proteins into target cells. Inhibitors targeting T3SS are important to combat the appearance of multidrug‐resistant pathogens. In Pseudomonas aeruginosa, the T3SS secreted proteins PopB (SctE) and PopD (SctB) are required to inject effector proteins into the host cells. PopB and PopD associate with the target membrane and they are proposed to form a translocon pore by which effectors are translocated. This translocon is a potential target for inhibitors because these molecules do not need to overcome the intrinsic problems associated with cell membrane permeability and the presence of efflux pumps.In previous studies, we have reported that the PopD N‐terminus is exposed to host cell cytosol (J. Biol. Chem. 2018 293 8982–8993). We reasoned that the PopD N‐terminal exposure could be used as a reporter for properly assembled translocons. Using a split form of the small NanoLuc (NLuc) luciferase, we have developed an assay to detect the exposure of this N‐terminus to the host cytosol. HeLa cells producing the large truncated version of NLuc are used as targets. These cells are incubated with a P. aeruginosa ΔpopD PAK strain that produces a PopD variant fused with the missing portion of NLuc at the N‐terminus (Nluc10‐PopD). Assembly of a functional translocon is detected by the maturation of NLuc enzyme in the HeLa cell cytosol and the production of luminescence. Failure to assemble a functional translocon did not produce luminescence. The assay has a typical signal‐to‐background ratio (S/B) of 6 ‐ 10 and a Z factor above 0.5, constituting an excellent tool for the screening and detection of small molecule inhibitors of T3SS translocon assembly.

Sulopenem: An Intravenous and Oral Penem for the Treatment of Urinary Tract Infections Due to Multidrug-Resistant Bacteria.

Sulopenem: An Intravenous and Oral Penem for the Treatment of Urinary Tract Infections Due to Multidrug-Resistant Bacteria.

Pseudomonas aeruginosa efflux pump superfamily (review of literature).

The significant increase in the number of antibiotic-resistant microorganisms observed in recent years is a public health problem worldwide. One of the molecular mechanisms for the formation of antimicrobial resistance in bacteria is the presence of efflux pumps. The review presents an analysis of experimental studies related to the study of efflux pumps in clinical strains of Pseudomonas aeruginosa, one of the representatives of hospital pathogens of the ESKAPE group. This review is intended for specialists developing new types of drugs against antibiotic-resistant strains, as well as researchers studying the mechanisms of bacterial resistance to antibiotics, heavy metals, biocides and other antimicrobial factors.

Antibiotic Resistance, Virulence Factors and Phylogenetic Analysis of Efflux Proteins of Coagulase Negative <i>Staphylococcus</i> Isolates from Sewage Samples

Multidrug-resistant pathogens are now emergent worldwide and pose a serious threat to disease prevention and treatment. The World Health Organization declared anti-microbial resistance as a major peril to human beings. Staphylococcus is part of the normal microbiome of humans and are responsible for opportunistic infections. Like S. aureus, Coagulase-negative staphylococcus are also clinically important as the causal agents of severe diseases, nosocomial infections, catheter-associated infection, bacteremia, septicemia. Methicillin resistant Staphylococci species are widely known and methicillin resistant CoNS, known as MR-CoNS, have been found to carry the mecA gene. Various virulence factors help these organisms in their pathogenicity and antibiotic-resistance is aided by the presence of efflux pumps. In our study, we have isolated 45 isolates from sewage water which were tentatively identified as CoNS based on biochemical characteristics. Then virulence and antibiotics susceptibility patterns were screened by standard protocols. Multiple drug resistant CoNS were found in our study and in silico analysis of an annotated protein sequence of the efflux pump SepA of Pseudomonas putida was carried out. Sequence analysis of the efflux protein gene revealed that it is phylogenetically related to the AcrA of Staphylococcus sp. and a RND transporter of Vibrio sp. The widespread presence of MR-CoNS is a cause for serious concern as sewage treatment plants are reservoirs for the spread of antibiotic resistance.

Microorganisms and cationic surfactants

Quaternary ammonium salts (QAS) as cationic surfactants with an amphiphilic structure show biocidal activity against non-pathogenic and pathogenic microorganisms (Gram-positive, Gram-negative bacteria, fungi, as well as lipophilic viruses) occurring in planktonic form or forming biofilms. They can also coat the surface of various materials (glass, metal, silicone, polyester) from which medical devices are made, such as: catheters, implants, heart valves, endoprostheses, and this allows inhibiting the adhesion of microorganisms to these surfaces. In their chemical structure, these surfactants contain labile bonds, e.g. ester or amide bonds, which enable the biodegradation of the compounds. Thanks to this, they are classified as “green chemistry”. Their biological activity depends on the length of the hydrophobic chain and the structure of the hydrophilic head of the compound. QAS have an affinity for the cell membrane by interacting with its lipids and proteins, which can lead to its disintegration. They have the ability to inhibit the activity of H+-ATPase of the cell membrane, the enzyme responsible for its electrochemical gradient and the transport of nutrients to the cell, e.g. amino acids. These compounds can influence the lipid composition (quantitative and qualitative) of the cell membrane of microorganisms. They are also inhibitors of respiratory processes and can induce the formation of reactive oxygen species. These surfactants are capable of forming micelles and liposomes in an aqueous environment. They are widely used in medicine (as disinfectants, DNA carriers in gene therapy), in industry and environmental protection (as biocides, preservatives) and in agriculture (as fungicides). The widespread use of QAS in many fields causes the growing resistance of microorganisms to these compounds. A common mechanism that generates reduced susceptibility to QAS is the presence of efflux pumps.

Styrylpyridinium Derivatives as New Potent Antifungal Drugs and Fluorescence Probes.

The incidence of Candida glabrata infections increases every year due to its higher resistance to commonly used antifungal drugs. We characterized the antifungal mechanism of action of eight new styrylpyridinium derivatives, with various N-alkyl chains (-C6H13, -C8H17, -C10H21, -C12H25) and different substituents, on C. glabrata strains differing in their drug resistance due to the presence or absence of two major drug-efflux pumps. We found that the tested styrylpyridinium compounds affected the growth of C. glabrata cells in a compound- and strain-dependent manner, and apparently they were substrates of CgCdr1 and CgCdr2 pumps. Further, we determined the impact of the tested compounds on plasma membrane integrity. The ability to cause damage to a plasma membrane depended on the compound, its concentration and the presence of efflux pumps, and corresponded well with the results of growth and survival tests. We also tested possible synergism with three types of known antifungal drugs. Though we did not observe any synergism with azole drugs, styrylpyridinium compounds 5 and 6 together with FK506 demonstrated excellent antifungal properties, whereas compounds 2, 3, 5, and 6 exhibited a significant synergistic effect in combination with terbinafine. Based on our results, derivatives 2 and 6 turned out to be the most promising antifungal drugs. Moreover, compound 6 was not only able to effectively permeabilize the yeast plasma membrane, but also exhibited significant synergism with FK506 and terbinafine. Finally, we also characterized the spectroscopic properties of the tested styrylpyridinium compounds. We measured their absorption and fluorescence spectra, determined their localization in yeast cells and found that their fluorescence characteristics differ from the properties of current commercial vacuolar styrylpyridinium markers and allow multi-color staining. Compounds 1, 3, 7, and 8 were able to accumulate in plasma and vacuolar membranes, and compounds 2, 5, and 6 stained the whole interior of dead cells. In summary, of the eight tested compounds, compound 6 is the most promising antifungal drug, compound 8, due to its minimal toxicity, is the best candidate for a new vacuolar-membrane probe or new benchmark substrate of C. glabrata Cdr pumps, and derivative 5 for a new vital dye.

Efflux pump inhibitory activity of biologically synthesized silver nanoparticles against multidrug-resistant Acinetobacter baumannii clinical isolates.

This study was carried out to investigate the possible efflux pump inhibitory activity of biologically synthesized silver nanoparticles (AgNPs) against multidrug-resistant (MDR) Acinetobacter baumannii isolates. In this study, the physicochemical characteristics of synthesized AgNPs were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectrophotometer (FTIR) methods. Subsequently, MDR A. baumannii isolates were recovered from clinical samples and the phenotypic cartwheel efflux assay and polymerase chain reaction (PCR) were used to elucidate the possible presence of efflux pump in MDR strains. After treatment of MDR strains with sub-minimum inhibitory concentration (MIC) concentration of AgNPs, the expression level of efflux pump genes was evaluated using a quantitative real-time PCR technique. The synthesized AgNPs had a spherical nanostructure, with mean size 38.89 nm, according to SEM and TEM data. XRD and FTIR results confirmed the synthesis of AgNPs. The results of PCR revealed that among 50 strains, 12 A. baumannii strains had efflux pump genes and the expression level of AdeA, AdeC, AdeS, AdeR, AdeI, AdeJ, and AdeK efflux pump genes was downregulated significantly after the treatment with AgNPs. In addition, the inhibitory effect of AgNPs on efflux pumps can be detected when the MIC of ethidium bromide (EtBr) with AgNPs is lower than that of EtBr alone. According to the results, the biologically synthesized AgNPs exhibit efflux pumps inhibitory activity, which may be one of the possible mechanisms of their antibacterial activity against MDR A. baumannii strains.

Evaluation of multidrug efflux pump expression in clinical isolates of Staphylococcus.aureus

BackgroundOne of the mechanisms of Staphylococcus aureus resistance to ciprofloxacin is the presence of efflux pumps. The aim of this study was to determine the frequency and expression of norA, norB and norC efflux pump genes in ciprofloxacin-resistant Staphylococcus aureus clinical isolates. Materials and methodsIn this study, 40 isolates of S. aureus were obtained from blood and urine samples. Antibiotic resistance pattern by disc diffusion method and the minimum inhibitory concentration (MIC) was evaluated. Identification and expression of nor efflux family genes (norA, norB, and norC) in ciprofloxacin-resistant isolates of S. aureus were determined using Real-Time PCR method. ResultsThe resistant to ciprofloxacin, Gentamicin, Cotrimoxazol, Erythromycin and Amoxicillin was 42.5%, 12.5%, 12.5%, 50% and 87.5%, respectively. All of ciprofloxacin-resistant isolates were positive for of norA, norB, and norC genes. The results of Real-Time PCR showed that all ciprofloxacin-resistant isolates are active in the case of efflux pumps. Isolates showed different expressions of the norA, norB and norC genes. Expression levels of norA and norB increased by 6.8 and 7.1 fold, respectively. ConclusionThe results indicated that nor efflux family genes were expressed in all ciprofloxacin-resistant isolates of S. aureus. Moreover, there is an association between the expression of the efflux pump genes and resistance to ciprofloxacin in S. aureus isolates.

Comparative Study between SmeDEF Multidrug Efflux Pump and CTX-M ESBL as Resistance Determinants in Nosocomial Isolates of Stenotrophomonas Maltophilia

Background: Stenotrophomonas maltophilia (S. maltophilia) is an important opportunistic pathogen that is usually associated with hospital acquired infections. Increased antibiotic resistance among those isolates is a real life threatening problem. Efflux pump and ESBL are two main causes of resistance in S. maltophilia. Objectives: The aims of this study were to isolate S. maltophilia from different sites of nosocomial infections among patients in Mansoura University Hospitals (MUHs), to determine multidrug resistant (MDR) isolates, to characterize those isolates as regarding presence of SmeDEF efflux pump and CTX-M ESBL and to compare the prevalence of both determinants as a cause of resistance in those isolates. Methodology: The study was conducted on 220 S. maltophilia isolates from nosocomialy infected patients which were subjected to antimicrobial susceptibility testing to determine the MDR isolates that were phenotypically tested for presence of efflux pump mediated antibiotic resistance and ESBL production by microdilution test and double disk synergy test respectively followed by detection of SmeDEF and CTX-M genes by PCR in the phenotypically positive isolates. Results: Antibiotic susceptibility testing of the 220 S. maltophilia isolates revealed that 179 (81.4%) of them were MDR, of which 43 (24%) were positive for efflux pump system and 147 (82.1%) were positive for ESBL production by phenotypic tests. PCR revealed that SmeDEF gene was detected in 38 (21.2%) of the MDR S. maltophilia isolates, where as CTX-M gene was found to be harbored by only 12 (6.7%) of those isolates. Conclusion: SmeDEF efflux pump was found to be a more prevalent cause of multiple antibiotic resistance in S. maltophilia nosocomial isolates than CTX-M ESBL; this may be helpful in improving the patient outcome if the exact cause of resistance is kept in mind during designation of the patient treatment regimen.

The Impact of Efflux Pump Inhibitors on the Activity of Selected Non-Antibiotic Medicinal Products against Gram-Negative Bacteria.

The potential role of non-antibiotic medicinal products in the treatment of multidrug-resistant Gram-negative bacteria has recently been investigated. It is highly likely that the presence of efflux pumps may be one of the reasons for the weak activity of non-antibiotics, as in the case of some non-steroidal anti-inflammatory drugs (NSAIDs), against Gram-negative rods. The activity of eight drugs of potential non-antibiotic activity, active substance standards, and relevant medicinal products were analysed with and without of efflux pump inhibitors against 180 strains of five Gram-negative rod species by minimum inhibitory concentration (MIC) value determination in the presence of 1 mM MgSO4. Furthermore, the influence of non-antibiotics on the susceptibility of clinical strains to quinolones with or without PAβN (Phe-Arg-β-naphthylamide) was investigated. The impacts of PAβN on the susceptibility of bacteria to non-antibiotics suggests that amitriptyline, alendronate, nicergoline, and ticlopidine are substrates of efflux pumps in Gram-negative rods. Amitriptyline/Amitriptylinum showed the highest direct antibacterial activity, with MICs ranging 100–800 mg/L against all studied species. Significant decreases in the MIC values of other active substances (acyclovir, atorvastatin, and famotidine) tested with pump inhibitors were not observed. The investigated non-antibiotic medicinal products did not alter the MICs of quinolones in the absence and in the presence of PAβN to the studied clinical strains of five groups of species.

Chlorhexidine bathing for the prevention of colonization and infection with multidrug-resistant microorganisms in a hematopoietic stem cell transplantation unit over a 9-year period: Impact on chlorhexidine susceptibility.

Health care associated infections (HAIs) are currently among the major challenges to the care of hematopoietic stem cell transplantation (HSCT) patients. The objective of the present study was to evaluate the impact of 2% chlorhexidine (CHG) bathing on the incidence of colonization and infection with vancomycin-resistant Enterococcus (VRE), multidrug-resistant (MDR) gram-negative pathogens, and to evaluate their CHG minimum inhibitory concentration (MIC) after the intervention.A quasi-experimental study with duration of 9 years was conducted. VRE colonization and infection, HAI rates, and MDR gram-negative infection were evaluated by interrupted time series analysis. The antibacterial susceptibility profile and mechanism of resistance to CHG were analyzed in both periods by the agar dilution method in the presence or absence of the efflux pump inhibitor carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and presence of efflux pumps (qacA/E, qacA, qacE, cepA, AdeA, AdeB, and AdeC) by polymerase chain reaction (PCR).The VRE colonization and infection rates were significantly reduced in the postintervention period (P = 0.001). However, gram-negative MDR rates in the unit increased in the last years of the study. The CHG MICs for VRE increased during the period of exposure to the antiseptic. A higher MIC at baseline period was observed in MDR gram-negative strains. The emergence of a monoclonal Pseudomonas aeruginosa clone was observed in the second period.Concluding, CHG bathing was efficient regarding VRE colonization and infection, whereas no similar results were found with MDR gram-negative bacteria.

Nano-ART and NeuroAIDS.

Human immunodeficiency virus (HIV) is a neurotropic virus that enters the central nervous system (CNS) early in the course of infection. Although highly active antiretroviral therapy (HAART) has resulted in remarkable decline in the morbidity and mortality in AIDS patients, controlling HIV infections still remains a global health priority. HIV access to the CNS serves as the natural viral preserve because most antiretroviral (ARV) drugs possess inadequate or zero delivery across the brain barriers. The structure of the blood-brain barrier (BBB), the presence of efflux pumps, and the expression of metabolic enzymes pose hurdles for ARV drug-brain entry. Thus, development of target-specific, effective, safe, and controllable drug delivery approach is an important health priority for global elimination of AIDS progression. Nanoformulations can circumvent the BBB to improve CNS-directed drug delivery by affecting such pumps and enzymes. Alternatively, they can be optimized to affect their size, shape, and protein and lipid coatings to facilitate drug uptake, release, and ingress across the barrier. Improved drug delivery to the CNS would affect pharmacokinetic and drug biodistribution properties. This review focuses on how nanotechnology can serve to improve the delivery of antiretroviral medicines, termed NanoART, across the BBB and affect the biodistribution and clinical benefit for NeuroAIDS.

Antibiotic resistance profiling of marine halophilic bacteria and haloarchaea

Drug resistance in microorganisms is an emerging phenomenon that poses challenges to public health and treatment. The reservoir of drug resistance genes found in the resistome of environmental isolates in aquatic saline systems has been augmenting continuously. The spread of drug resistance is attributed to the dissemination of drug resistant pathogens occurring in sewage and wastewater released in the marine environments like seas. Halophiles are salt loving organisms that are found naturally in these marine environments, sea water, salterns and lakes. The present study aimed at investigation of the antibiotic resistance profile of halophiles isolated from marine ecosystems of the coastal Maharashtra, India using disk diffusion method. The plausible mechanism of drug resistance in the marine halophiles was investigated by detecting plasmids and antibiotic efflux pumps using EtBr-Cart wheel assay. The haloarchaea, Haloarcula sp. RR14 and Halovivax sp. RT5 showed multidrug resistance and presence of efflux pumps (MICEtBr= 0.5 µg/ml). The resistance of gram positive halophiles was in following order: Salimicrobium salexigens RR5>Salimicrobium flavidumRR2>Alkalibacillus almallahensis RR3. Amongst gram negative halophiles, their resistance was in following order: Halomonas smyrnensis RR7>Halomonas koreensis RR1>Marinobacter oulmenensis RR6. Plasmids were detected in Haloarcula sp., Halomonas sp. and Marinobacter sp. The present study related to antibiotic resistance of halophiles from marine environment is significant as some of the marine halophilic isolates like Halomonas sp. have been reported as potential pathogens in recent times.

Nanomedicine and its application in treatment of microglia-mediated neuroinflammation.

Nanomedicine, an emerging therapeutic tool in current medical frontiers, offers targeted drug delivery for many neurodegenerative disorders. Neuroinflammation, a hallmark of many neurodegenerative disorders, is mediated by microglia, the resident immunocompetent cells of the central nervous system (CNS). Microglial cells respond to various stimuli in the CNS resulting in their activation which may have a beneficial or a detrimental effect. In general, the activated microglia remove damaged neurons and infectious agents by phagocytosis, therefore being neuroprotective. However, their chronic activation exacerbates neuronal damage through excessive release of proinflammatory cytokines, chemokines and other inflammatory mediators which contribute to neuroinflammation and subsequent neurodegeneration in the CNS. Hence, controlling microglial inflammatory response and their proliferation has been considered as an important aspect in treating neurodegenerative disorders. Regulatory factors that control microglial activation and proliferation also play an important role in microglia-mediated neuroinflammation and neurotoxicity. Various anti-inflammatory drugs and herbal compounds have been identified in treating microglia-mediated neuroinflammation in the CNS. However, hurdles in crossing blood brain barrier (BBB), expression of metabolic enzymes, presence of efflux pumps and several other factors prevent the entry of these drugs into the CNS. Use of non-degradable delivery systems and microglial activation in response to the drug delivery system further complicate drug delivery to the CNS. Nanomedicine, a nanoparticle-mediated drug delivery system, exhibits immense potential to overcome these hurdles in drug delivery to the CNS enabling new alternatives with significant promises in revolutionising the field of neurodegenerative disease therapy. This review attempts to summarise various regulatory factors in microglia, existing therapeutic strategies in controlling microglial activation, and how nanotechnology can serve to improve the delivery of therapeutic drugs across the BBB for treating microglia- mediated neuroinflammation and neurodegeneration.

Polymorphism in gyrA is associated to quinolones resistance in Chilean Piscirickettsia salmonis field isolates.

Keywords: antibiotic resistance, gyrA, Piscirickettsiasalmonis, quinolones.Piscirickettsia salmonis is a gram-negative, pleomor-phic coccoid-shaped and facultative intracellularfish pathogenic bacterium, that causes piscirickettsi-osis, a disease also known as salmonid rickettsialsepticaemia (SRS) (Fryer et al. 1990). In Chile,P. salmonis was isolated for the first time in 1989,but probably has been present since 1983 (Bravo C Rozas &Enriquez 2013). SRS has also been reported inimportant salmon producing countries such asCanada, Ireland, Scotland and Norway, but with alower economic impact (Rozas & Enriquez 2013).P. salmonis cells are slow growing and hard to culti-vate in free-cell media, which has hindered its isola-tion and further characterization. However, recentlydescribed supplemented media (Mikalsen et al.2008) facilitate their growth and the antibiotictesting assays to evaluate effective antimicrobial ther-apy. In this regard, quinolones were the former mostwidely used antimicrobial compounds in Chileanaquaculture, especially as a first line drug againstSRS.In Chile, a large proportion of antibiotics is usedfor prophylaxis rather than for chemotherapy. Forinstance, in comparison with Norway and Canada,1500 and eight times more antimicrobials wereneeded to obtain similar amounts of salmon inChile in 2007 (Cabello et al. 2013). The same year,930 metric tons of antibiotics were mostly used insalmon production. Of those antibiotics, 24% werequinolones (Cabello et al. 2013). This practice hasalready altered the microbiota of sediments in closeproximity to aquaculture sites, where a significantfraction of isolated bacteria is antibiotic resistant(Buschmann et al. 2012). Therefore, the emer-gence and potential spread of antibiotic-resistantP. salmonis isolates cannot be ruled out.Twenty P. salmonis isolates cultured from kid-ney samples of diseased fish collected from diversegeographic origin (Fig. 1) between 2010 and2012 were phenotypically characterized for quinol-ones susceptibility. The isolates were grown onagar (Mikalsen et al. 2008), at 18 °C for 10 daysand kept frozen in liquid nitrogen. For antibioticsensitivity testing, the minimal inhibitory concen-tration (MIC) for enrofloxacin, flumequine andoxolinic acid were assessed according to theinstructions given by the Clinical and LaboratoryStandards Institute (CLSI), guide M49-A. 96-wellmicroplates containing twofold serial dilutions(0.03–64 lgmL

Caracterização fenotípica, produção de biofilme e resistência aos antimicrobianos em isolados de Staphylococcus spp. obtidos de casos de mastite em bovinos e bubalinos

No presente estudo, objetivou-se avaliar a sussuscetibilidade aos principais antimicrobianos e realizar uma caracterização fenotípica e genotípica de isolados de Staphylococcus spp. obtidos de casos de mastite em vacas (n=30) e búfalas (n=30). A suscetibilidade foi avaliada pela técnica de disco-difusão e a presença de bomba de efluxo foi avaliada utilizando-se Ágar Mueller Hinton (MH) adicionado de brometo de etídeo e pesquisa do gene msrA. Pela técnica da Reação em Cadeia da Polimerase (PCR) ainda foram identificados os genes mecA, blaZ e ermA, B e C, que posteriormente foram associados com os métodos fenotípicos para a identificação de resistência a antimicrobianos. A caracterização da formação de biofilme foi realizada utilizandose os métodos Ágar Vermelho Congo (CRA), Aderência em Placa e a identificação do gene icaD. Pelo método de discodifusão, os Staphylococcus spp. apresentaram alta sensibilidade aos antimicrobianos. O índice de resistência múltipla aos antimicrobianos (IRMA) apresentou variação de 0 a 0,5. Na pesquisa de bomba de efluxo, 26,7% das amostras foram positivas ao método fenotípico e 6,7% ao método genotípico (gene msrA). Os genes erm, mecA e blaZ foram detectados, respectivamente, em 1,7%, 6,7% e 11,7% das amostras de Staphylococcus spp. Na produção de biofilme, 23,3% dos isolados foram considerados positivos no CRA, 50,0% na Aderência em Placas e 8,3% na PCR pela detecção do gene icaD. Observou-se que os isolados obtidos de amostras bovinas apresentaram uma menor sensibilidade aos antimicrobianos no teste de disco-difusão quando comparados com as amostras bubalinas. A caracterização destes isolados é importante para orientar uma antibioticoterapia bem planejada. A presença de biofilme nos isolados pode estar associada a outros fatores que não a resistência às drogas antimicrobianas.

Accumulation and distribution of doxorubicin in tumour spheroids: the influence of acidity and expression of P-glycoprotein

The intra-tumour distribution of anticancer drugs remains an important, but often under-estimated, influence on drug efficacy. Tumour acidity and the presence of efflux pumps were examined for their influence on the distribution of doxorubicin in a solid tumour model. Anticancer drug distribution and overall accumulation was measured in tumour spheroids (TS) of varying sizes. The distribution profiles were examined in normoxic and hypoxic TS, the latter generating metabolic acidosis. Finally, the drug distribution profiles were related to efficacy using radial outgrowth assays. In large tumour spheroids (TS) (d ~500μm), intracellular accumulation of doxorubicin was restricted to cells in the outermost layers and failed to accumulate within the viable cells in the 'intermediate' hypoxic zone. A similar profile was obtained for another protonatable amine, 7-AAD. In contrast, the distribution of the non-ionisable drug (at physiological pH) BODIPY-Taxol was uniform throughout the TS. In order to independently model the hypoxic and normoxic zones of TS, we compared drug accumulation in small entirely normoxic TS (d ~200μm) with equivalent sized ones exposed to hypoxia in an anaerobic chamber. Exposure of TS to hypoxia caused a considerable reduction in the pH of the bathing medium and lower tissue accumulation of doxorubicin. Interstitial acidity reduces the proportion of doxorubicin in the non-ionised form. In TS, the accumulation and distribution of doxorubicin was influenced by both the expression of P-glycoprotein and hypoxia-induced acidity. Therefore, optimisation of doxorubicin chemotherapy for hypoxic tumours will require circumvention of both of these crucial pharmacokinetic determinants.

NanoART, NeuroAIDS and CNS Drug Delivery

A broad range of nanomedicines is being developed to improve drug delivery for CNS disorders. The structure of the blood-brain barrier (BBB), the presence of efflux pumps and the expression of metabolic enzymes pose hurdles for drug-brain entry. Nanoformulations can circumvent the BBB to improve CNS-directed drug delivery by affecting such pumps and enzymes. Alternatively, they can be optimized to affect their size, shape, and protein and lipid coatings to facilitate drug uptake, release and ingress across the barrier. This is important as the brain is a sanctuary for a broad range of pathogens including HIV-1. Improved drug delivery to the CNS would affect pharmacokinetic and drug biodistribution properties. This article focuses on how nanotechnology can serve to improve the delivery of antiretroviral medicines, termed nanoART, across the BBB and affect the biodistribution and clinical benefit for HIV-1 disease.