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An increase in Klebsiella pneumoniae carbapenem-resistant human nosocomial strains is occurring in Europe, namely with the bla OXA-48-like and bla KPC-like genes. We determined the prevalence of carbapenemase-producing Enterobacterales clinical strains in companion animals in Portugal and characterized their mobile genetic elements. Susceptibility data of a consecutive collection of 977 Enterobacterales clinical strains from a Portuguese private veterinary diagnostic laboratory were evaluated (January-December 2020). Additional phenotypical and genotypical assays were performed in a subset of 261 strains with a resistant phenotype. Whole-genome sequencing was performed for carbapenemase-producing strains. The frequency of carbapenemase-producing Enterobacterales clinical strains in companion animals in Portugal was 0.51% ( n = 5/977). Thus, five strains were characterized: (i) one OXA-181-producing K. pneumoniae ST273, (ii) two KPC-3-producing K. pneumoniae ST147; (iii) one KPC-3-producing K. pneumoniae ST392; and (iv) one OXA-48-producing E. coli ST127. The bla KPC-3 gene was located on transposon Tn 4401d on IncFIA type plasmid for the K. pneumoniae ST147 strains and on a IncN-type plasmid for the K. pneumoniae ST392 strain, while bla OXA-181 gene was located on an IncX3 plasmid. All de novo assembled plasmids and plasmid-encoded transposons harboring carbapenemase genes were homologous to those previously described in the human healthcare. No plasmid replicons were detected on the OXA-48-producing E. coli ST127. The dissemination of carbapenem resistance is occurring horizontally via plasmid spreading from the human high burden carbapenem resistance setting to the companion animal sector. Furthermore, companion animals may act as reservoirs of carbapenem resistance. Implementation of carbapenemase detection methods in routine clinical veterinary microbiology is urgently needed., Importance: This is the first study on the prevalence of carbapenemase-producing Enterobacterales (CPE) clinical strains from companion animals in Portugal. Despite the generally low prevalence of CPE in companion animals, it is imperative for veterinary diagnostic laboratories to employ diagnostic methods for carbapenemase detection. The resemblance found in the mobile genetic elements transporting carbapenemase genes between veterinary medicine and human medicine implies a potential circulation within a One Health framework., Competing Interests: The authors declare no conflict of interest.

The temperature dependence of the electrical properties of composites formed by biphasic sodium titanate and poly( o -methoxyaniline) (Na 2 Ti 3 O 7 /Na 2 Ti 6 O 13 /POMA) with different concentrations of POMA (0%, 1%, 10%, 15%, 35% and 50%) in the ceramic matrix was determined from measurements of complex impedance. The structural details were studied by means of X-ray diffraction, confirming the formation of the Na 2 Ti 3 O 7 /Na 2 Ti 6 O 13 /POMA composites. The displacement of the (200) reflection from 2θ = 10.45° to 11.15° in the composites with 10 and 15% of POMA suggested the partial replacement of H + for Na + in the Na 2 Ti 3 O 7 structure. The thermal properties were investigated by Thermogravimetry and Differential Thermal Analysis. The Thermogravimetry curves of the composites with POMA content of 1, 10 and 15% presented profiles similar to that of pure sodium titanate sample. The composites with 35 and 50% of POMA showed a process at temperatures around 60-70 °C, which was associated with water absorbed by the polymer. The analysis of the complex impedance spectroscopy measurements revealed that the electrical resistivity of the composites in the range from 0 to 35% increased by two orders of magnitude, with different values for each concentration. This positive temperature coefficient of resistivity was less noticeable in the composite with highest POMA mass content (50%). The rapid increase in resistivity caused an increase in the relaxation time calculated from the time domain. The electrical response of the 50% of POMA compound changes in relation to what was observed in the other compounds, which suggests that there is a saturation limit in the increase in resistivity with POMA content.

Antibiotic resistance became an increasing risk for population health threatening our ability to fight infectious diseases. The objective of this study was to evaluate the activity of laser irradiated thioridazine (TZ) against clinically-relevant bacteria in view to fight antibiotic resistance. TZ in ultrapure water solutions was irradiated (1-240 min) with 266 nm pulsed laser radiation. Irradiated solutions were characterized by UV-Vis and FTIR absorption spectroscopy, thin layer chromatography, laser-induced fluorescence, and dynamic surface tension measurements. Molecular docking studies were made to evaluate the molecular mechanisms of photoproducts action against Staphylococcus aureus and MRSA. More general, solutions were evaluated for their antimicrobial and efflux inhibitory activity against a panel of bacteria of clinical relevance. We observed an enhanced antimicrobial activity of TZ photoproducts against Gram-positive bacteria. This was higher than ciprofloxacin effects for methicillin- and ciprofloxacin-resistant Staphylococcus aureus. Molecular docking showed the Penicillin-binding proteins PBP3 and PBP2a inhibition by sulforidazine as a possible mechanism of action against Staphylococcus aureus and MRSA strains, respectively. Irradiated TZ reveals possible advantages in the treatment of infectious diseases produced by antibiotic-resistant Gram-positive bacteria. TZ repurposing and its photoproducts, obtained by laser irradiation, show accelerated and low-costs of development if compared to chemical synthesis.

Background: Antimicrobial resistance is a problem in human and animal healthcare. Honey may be used for its wound healing properties and antimicrobial effects., Objective: To investigate the antimicrobial activity of two commercially available medical grade honeys (MGHs) against Staphylococcus spp. and Pseudomonas spp. isolates., Methods and Materials: Two formulations, MGH1 (40% w/v honey) and MGH2 (80% w/v Manuka honey), were tested in vitro for minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) against 11 Staphylococcus and 11 Pseudomonas isolates at low [1.5 × 10 4  colony forming units (cfu)/well] and high (1.5 × 10 6  cfu/well) concentrations of inoculum, representing systemic and cutaneous bacterial loads during infection, respectively., Results: MGH2 showed a lower MIC against staphylococci than MGH1, although this was not statistically significant. MGH1 had stronger bactericidal effects against staphylococci than MGH2, although this effect was statistically significant only at the higher bacterial concentration (P < 0.01). For Pseudomonas spp., MGH1 had significantly higher antimicrobial activity (both MIC and MBC) than MGH2 against all isolates tested and at both bacterial concentrations (P < 0.05)., Conclusions and Clinical Importance: Both MGHs were effective in vitro against common cutaneous pathogens including meticillin-resistant staphylococci and Pseudomonas species. The higher efficacy of the MGH1 formulation against Pseudomonas and its consistent effects against staphylococci, while containing only half of the amount of honey compared to MGH2, invites further investigation of the mechanisms and clinical applications of MGH1., (© 2019 ESVD and ACVD.)

Mycobacterium avium is a difficult-to-treat pathogen able to quickly develop drug resistance. Like for other microbial species, overexpression of efflux pumps is one of the main mechanisms in developing multidrug resistance. Although the use of efflux pumps inhibitors (EPIs) represents a promising strategy to reverse resistance, to date few M. avium EPIs are known. Recently, we showed that in-house 2-phenylquinoline S. aureus NorA EPIs exhibited also a good activity against M. avium efflux pumps. Herein, we report a series of 3-phenylquinolones designed by modifying the isoflavone biochanin A, a natural EPI of the related M. smegmatis, taking into account some important SAR information obtained around the 2-phenylquinoline NorA EPIs. The 3-phenylquinolones inhibited M. avium efflux pumps with derivatives 1e and 1g that displayed the highest synergistic activity against all the strains considered in the study, bringing down (from 4- to 128-fold reduction) the MIC values of macrolides and fluoroquinolones., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Objectives: The aim of this study was to characterize 32 MRSA isolates recovered from wound specimens of patients in a Hospital in Tripoli, Libya, during 2013., Methods: MRSA isolates were characterized by determining their antibiotic susceptibilities, genes encoding antibiotic resistance and virulence factors, the SCCmec class, agr type, spa typing, PFGE and MLST., Results: PFGE and MLST revealed that all isolates were clonal and belonged to the Clonal Complex 5 (CC5). They harboured the SCCmecV and the agrII and the spa type was t6065. The majority of isolates were resistant to cefoxitin (32, 100%), penicillin (32, 100%), ampicillin (32, 100%), enrofloxacin (32, 100%), ciprofloxacin (32, 100%), fusidic acid (32, 100%), gentamicin (32, 100%), kanamycin (32, 100%), trimethoprim (32, 100%), and erythromycin (30, 93.7%). The main genes encoding antibiotic resistance were: blaZ (31, 96.8%), ermC (30, 93.7%), aph(3')-III a (3, 9.4%), aac6-aph2 (32, 100%), InuA (3, 9.4%), tetM (3, 9.4%), tetL (3, 9.4%), dfrG (28, 87.5%), fusC (32, 100%). All isolates were PVL negative; however, exfoliative-encoding genes (eta: 25) and enterotxin genes (seb: 32, seo: 32, sei: 32, ser: 32, seu: 32, seg: 32, sej: 32, sed: 31, sen: 29, seh: 26, sec: 26, sea: 6, sek: 5), haemolysin (hla (32), hld (32), hlg (32)) and immune evasion cluster proteins (scn: 32, sak: 32) were relevant., Conclusion: To the best of our knowledge this is the first report of a specific clonal spread of a multi-drug resistant MRSA-CC5- SCCmecV in a Libyan Hospital., (Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Objective: To evaluate whether hypoxia inducible factor (HIF-1α) targeting pharmacological drugs, echinomycin, resveratrol and CdCl2 which inhibit HIF-1α stimulation, and mimosine, which enhances the stability of HIF-1α present antileishmanial properties., Methods: The leishmanicidal effect of drugs was evaluated in mouse macrophages and Balb/c mouse model for cutaneous leishmaniosis., Results: Resveratrol and CdCl2 reduced the parasite load [IC50, (27.3 ± 2.25) μM and (24.8 ± 0.95) μM, respectively]. The IC50 value of echinomycin was (22.7 ± 7.36) nM and mimosine did not alter the parasite load in primary macrophages. The macrophage viability IC50 values for resveratrol, echinomycin and CdCl2 and mimosine were >40 μM, >100 nM, >200 μM and>2000 μM, respectively. In vivo no differences between cutaneous lesions from control, resveratrol- and echinomycin-treated Balb/c mice were detected., Conclusions: Resveratrol, echinomycin and CdCl2 reduce parasite survival in vitro. The HIF-1α targeting pharmacological drugs require further study to more fully determine their anti-Leishmania potential and their role in therapeutic strategies., (Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.)

The identification of efflux inhibitors to be used as adjuvants alongside existing drug regimens could have a tremendous value in the treatment of any mycobacterial infection. Here, we investigated the ability of four 2-(4'-propoxyphenyl)quinoline Staphylococcus aureus NorA efflux inhibitors (1-4) to reduce the efflux activity in Mycobacterium smegmatis and Mycobacterium avium strains. All four compounds were able to inhibit efflux pumps in both mycobacterial species; in particular, O-ethylpiperazinyl derivative 2 showed an efflux inhibitory activity comparable to that of verapamil, the most potent mycobacterial efflux inhibitor reported to date, and was able to significantly reduce the MIC values of macrolides against different M. avium strains. The contribution of the M. avium efflux pumps MAV&#95;1406 and MAV&#95;1695 to clarithromycin resistance was proved because they were found to be overexpressed in two M. avium 104 isogenic strains showing high-level clarithromycin resistance. These results indicated a correlation between increased expression of efflux pumps, increased efflux, macrolide resistance, and reduction of resistance by efflux pump inhibitors such as compound 2. Additionally, compound 2 showed synergistic activity with clarithromycin, at a concentration below the cytotoxicity threshold, in an ex vivo experiment against M. avium 104-infected macrophages. In summary, the 2-(4'-propoxyphenyl)quinoline scaffold is suitable to obtain compounds endowed with good efflux pump inhibitory activity against both S. aureus and nontuberculous mycobacteria.

Background: Efflux has been recognized as a resistance mechanism to antimicrobials in Staphylococcus aureus; however its role on the development of clinically relevant resistance is still poorly characterized. This study aimed to examine the impact of efflux on development of resistance to fluoroquinolones and other antimicrobials in S. aureus strains representing relevant phenotypes in terms of antibiotic susceptibility and efflux activity., Methods: Two closely related methicillin- and ciprofloxacin-resistant Staphylococcus aureus clinical strains, with different efflux capacity and the pan-susceptible strain ATCC25923 were exposed to constant concentrations of the efflux pump (EP) substrates ciprofloxacin, ethidium bromide and cetrimide. Parental and exposed strains were tested regarding their susceptibility towards antibiotics, biocides and ethidium bromide, efflux capacity and levels of EP gene expression. Occurrence of resistance-associated mutations was screened by sequencing., Results: Multidrug resistance phenotypes emerged upon exposure, independently of the substrate or its concentration, which were correlated with increased efflux capacity of the exposed strains. The temporal pattern of EP gene expression disclosed an early-response with high expression of several genes, followed by a late-response, characterized by overexpression of specific genes. The overall cell response was more pronounced for strains with an initial basal efflux activity. Remarkably, detection of the IS256 element in the promoter regions of mgrA and norA, in some cases associated with increased gene expression, suggests that these genes may be hot spots for IS256 insertion events. The results obtained with exposure of ATCC25923 to ciprofloxacin were particularly striking, revealing a step-wise development of fluoroquinolone resistance, with a first efflux-mediated response, followed by the occurrence of a mutation in grlA that resulted in phenotypic resistance. Additionally, challenge by non-fluoroquinolone agents, particularly cetrimide, promoted cross resistance to fluoroquinolones, revealing the potential role of biocides as selective pressure for the emergence of resistance to these antibiotics., Conclusions: This study reveals efflux as a significant component of S. aureus resistance to fluoroquinolones and biocides and as a primary mechanism to withstand stress imposed by antimicrobials. This efflux-mediated response can result in the emergence of multidrug resistance in healthcare environments and should be taken into account in the management of this major pathogen.

Tuberculosis, caused by Mycobacterium tuberculosis, is still one of the leading infectious diseases globally. Therefore, novel approaches are needed to face this disease. Efflux pumps are known to contribute to the emergence of M. tuberculosis drug resistance. Thioridazine has shown good anti-TB properties both in vitro and in vivo, likely due to its capacity to inhibit efflux mechanisms. Here we report the design and synthesis of a number of putative efflux inhibitors inspired by the structure of thioridazine. Compounds were evaluated for their in vitro and ex vivo activity against M. tuberculosis H37Rv. Compared to the parent molecule, some of the compounds synthesized showed higher efflux inhibitory capacity, less cytotoxicity, and a remarkable synergistic effect with anti-TB drugs both in vitro and in human macrophages, demonstrating their potential to be used as coadjuvants for the treatment of tuberculosis.

Chemotherapy for leishmaniosis a neglected parasitic disease, is based on few drugs, which are toxic and present resistance issues. Efforts for the development of new therapies are essential for the control of leishmaniasis. Metabolic pathway enzymes are promising targets for new drugs against parasites. The search for effective drugs against key enzymes can take advantage of the similarities between metabolic pathways in different microorganisms trypanosomatids Trypanosoma cruzi and Leishmania and fungus Saccharomyces cerevisiae. In this report, leishmanicidal activity of the metabolic pathway enzymes inhibitors (IDs) of dihydroorotate dehydrogenase (DHODH), glyceraldehyde 3-phosphate dehydrogenase and cruzain-cysteine protease from T. cruzi and scitalona-desidratase, adenosine deaminase, succinate dehydrogenase complex II and hydroxynaphthalene reductase from S. cerevisiae was performed on Leishmania amazonensis extracellular promastigotes and amastigotes within macrophages. The most promising compound, ID195, which is a DHODH inhibitor was toxic against promastigotes and was selective for amastigotes over host cells., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Background: There are many techniques that can be used to reconstruct anomalous breast volume in Poland's syndrome, but repair of the stigmatizing deformities such as the transverse skin fold in the anterior axillary pillar, infraclavicular depression, and anomalous breast contours continues to be a challenge. This study aimed to demonstrate the superior results of laparoscopically harvested omentum flap to achieve these aesthetic improvements., Methods: Patients with Poland's syndrome from a clinical database were identified and their outcomes were studied., Results: In 15 consecutive patients with Poland's syndrome, the breast contour, the anterior axillary pillar, and the infraclavicular depression were treated with omentum flap and evaluated. Silicone implants were used beneath the flap in 80% of cases to improve symmetry. Flap consistency was similar to that of the natural breast and only a small incision in the breast fold was needed. The flap is extremely malleable, adapts to irregular surfaces, and has a long vascular pedicle. It does not leave a scar at the donor site as muscular flaps do. The omentum can repair small irregularities in breast contour, achieving a natural result different from all other flaps. Due to its malleability, it is possible to reconstruct even the extension to the axillary pillar, which is impossible with all other techniques., Conclusions: The omentum flap technique is a means of repairing the deformities caused by Poland's syndrome and improves the aesthetic result with outcomes that seem superior to any other reconstructive option.