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We investigated the in vitro antibacterial activity of the combination rifampicin (RIF) + polymyxin B (PB) against extensively drug-resistant (XDR) Klebsiella pneumoniae isolates. We evaluated clinical isolates co-resistant to PB (non-mcr carriers; eptB, mgrB, pmr operon, and ramA mutations) and to carbapenems (KPC, CTX-M, and SHV producers; including KPC + NDM co-producer), belonging to sequence types (ST) ST16, ST11, ST258, ST340, and ST437. We used the standard broth microdilution method to determine RIF and PB minimum inhibitory concentration (MIC) and the checkerboard assay to evaluate the fractional inhibitory concentration index (FICI) of RIF + PB as well as to investigate the lowest concentrations of RIF and PB that combined (RIF + PB) had antibacterial activity. Time-kill assays were performed to evaluate the synergistic effect of the combination against selected isolates. PB MIC (32-256 µg/mL) and RIF MIC (32-1024 µg/mL) were determined. FICI (<0.5) indicated a synergistic effect for all isolates evaluated for the combination RIF + PB. Our results showed that low concentrations of PB (PB minimal effective antibiotic concentration [MEAC], ≤0.25-1 µg/mL) favor RIF (≤0.03-0.125 µg/mL) to reach the bacterial target and exert antibacterial activity against PB-resistant isolates, and the synergistic effect was also observed in time-kill results. The combination of RIF + PB showed in vitro antibacterial activity against XDR, carbapenem-, and PB-resistant K. pneumoniae and could be further studied as a potential combination therapy, with cost-effectiveness and promising efficacy.

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Klebsiella quasipneumoniae subsp. similipneumoniae has emerged as a human pathogen and sporadic isolates from non-clinical sources were reported. Here, we described the phenotypic- and genomic-characteristics of a multidrug-resistant (MDR) and potentially hypervirulent (MDR-hv) Klebsiella quasipneumoniae subsp. similipneumoniae (KqA1) isolated from hospital wastewater. The antibiotic susceptibility profile of KqA1 was investigated using disk-diffusion method, broth microdilution method, and agar dilution method, and the genetic characteristics of antimicrobial resistance, mobile genetics elements, and virulence were evaluated by genomic DNA sequencing on the Illumina® NovaSeq6000 platform as well as by bioinformatic analysis. Resistome analyses revealed the presence of genes related to resistance to ß-lactams, aminoglycosides, quinolones, tetracyclines, sulfonamides, trimethoprim, chloramphenicol, macrolides, and fosfomycin. New genetic contexts to blaGES-16 (carbapenemase gene) and to fosA (fosfomycin resistance gene) were described. A set of mechanisms that can contribute to antibiotic resistance, commonly detected in Klebsiella spp., was also found including chromosomal mutations, efflux systems, proteins, and regulators. Moreover, KqA1 presented genes related to tolerance to metals (arsenic, copper, nickel, cobalt, magnesium, cadmium, zinc, tellurium, selenium) and to biocides (quaternary-ammonium compounds). The isolate was classified as potentially hypervirulent due to a wide range of virulence factors found associated to regulation, motility, biofilm, effector delivery systems, immune modulation, nutritional/metabolic factors, adherence, invasion, and competitive advantage. The occurrence of MDR-hv KqA1 in hospital wastewater points out how this environment matrix plays a crucial role in the maintenance and selection of critical bacterial pathogens. Regarding One Health perspective, it is evident the need for multidisciplinary implementation of control measures for antibiotic-resistant bacteria, not only in hospital settings but also in a general environmental context to mitigate the dissemination of MDR and hv bacteria.

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We correlated clinical, epidemiological, microbiological, and genomic data of an outbreak with polymyxin B (PB)- and carbapenem-resistant Klebsiella pneumoniae during the COVID-19 pandemic. Twenty-six PB- and carbapenem-resistant K. pneumoniae were isolated from patients in the COVID-19 ICU (Intensive Care Unit), non-COVID-19 ICU (Intensive Care Unit), clinical, or surgical ward. Bacterial identification, drug susceptibility tests, and DNA sequencing were performed, followed by in silico resistance genes identification. All isolates showed extensively drug-resistant (XDR) phenotypes. Four different sequence types (ST) were detected: ST16, ST11, ST258, and ST437. Nineteen isolates were responsible for an outbreak in the ICU in September 2020. They belong to ST258 and harbored the 42Kb IncX3plasmid (pKP98M3N42) with the same genomic pattern of two K. pneumoniae identified in 2018. Twenty-four isolates carried bla-KPC-2 gene. No plasmid-mediated colistin (mcr) resistance genes were found. Eight isolates presented mgrB gene mutation. The clonal isolates responsible for the outbreak came from patients submitted to pronation, with high mortality rates in one month. XDR-K. pneumoniae detected during the outbreak presented chromosomal resistance to PB and plasmid-acquired carbapenem resistance due to KPC production in most isolates and 42Kb IncX3(pKP98M3N42) plasmid carrying blaKPC-2 was associated with ST258 isolates. The outbreak followed the collapse of the local healthcare system with high mortality rates.

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This study aimed to assess the ultrapure cannabidiol (CBD) antibacterial activity and to investigate the antibacterial activity of the combination CBD + polymyxin B (PB) against Gram-negative (GN) bacteria, including PB-resistant Gram-negative bacilli (GNB). We used the standard broth microdilution method, checkerboard assay, and time-kill assay. CBD exhibited antibacterial activity against Gram-positive bacteria, lipooligosaccharide (LOS)-expressing GN diplococcus (GND) (Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis), and Mycobacterium tuberculosis, but not against GNB. For most of the GNB studied, our results showed that low concentrations of PB (≤ 2 µg/mL) allow CBD (≤ 4 µg/mL) to exert antibacterial activity against GNB (e.g., Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii), including PB-resistant GNB. CBD + PB also showed additive and/or synergistic effect against LOS-expressing GND. Time-kill assays results showed that the combination CBD + PB leads to a greater reduction in the number of colony forming units per milliliter compared to CBD and PB alone, at the same concentration used in combination, and the combination CBD + PB was synergistic for all four PB-resistant K. pneumoniae isolates evaluated. Our results show that CBD has translational potential and should be further explored as a repurposed antibacterial agent in clinical trials. The antibacterial efficacy of the combination CBD + PB against multidrug-resistant and extensively drug-resistant GNB, especially PB-resistant K. pneumoniae, is particularly promising.

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Neospora caninum is an apicomplexan parasite that causes abortion in cattle, resulting in significant economic losses. There is no commercial treatment for neosporosis, and drug repositioning is a fast strategy to test possible candidates against N. caninum. In this article, we describe the effects of atovaquone, chloroquine, quinine, primaquine and tetracycline on N. caninum proliferation. The IC50 concentrations in N. caninum were compared to the current information based on previous studies for Plasmodium and Toxoplasma gondii, correlating to the described mechanisms of action of each tested drug. The inhibitory patterns indicate similarities and differences among N. caninum, Plasmodium and T. gondii. For example, atovaquone demonstrates high antiparasitic activity in all the analyzed models, while chloroquine does not inhibit N. caninum. On the other hand, tetracycline is effective against Plasmodium and N. caninum, despite its low activity in T. gondii models. The repurposing of antimalarial drugs in N. caninum is a fast and inexpensive way to develop novel formulations using well-established compounds.

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Neospora caninum is an apicomplexan parasite that causes abortion in cattle, resulting in significant economic losses. There is no commercial treatment for neosporosis, and drug repositioning is a fast strategy to test possible candidates against N. caninum. In this article, we describe the effects of atovaquone, chloroquine, quinine, primaquine and tetracycline on N. caninum proliferation. The IC50 concentrations in N. caninum were compared to the current information based on previous studies for Plasmodium and Toxoplasma gondii, correlating to the described mechanisms of action of each tested drug. The inhibitory patterns indicate similarities and differences among N. caninum, Plasmodium and T. gondii. For example, atovaquone demonstrates high antiparasitic activity in all the analyzed models, while chloroquine does not inhibit N. caninum. On the other hand, tetracycline is effective against Plasmodium and N. caninum, despite its low activity in T. gondii models. The repurposing of antimalarial drugs in N. caninum is a fast and inexpensive way to develop novel formulations using well-established compounds.(AU)

Neospora caninum é um parasita Apicomplexa relacionado a abortos no gado bovino, que resultam em impactos econômicos. Não há tratamento comercial para neosporosis e o reposicionamento de drogas indica uma estratégia rápida para testar candidatos anti-N. caninum. Neste artigo, são descritos os efeitos da atovaquona, cloroquina, quinino, primaquine e tetraciclina na proliferação de N. caninum. As concentrações IC50 em N. caninum foram comparadas com a informação disponível, baseada em estudos publicados previamente para Plasmodium e Toxoplasma gondii, incluindo a correlação com os mecanismos de ação descritos para cada droga testada. Os padrões de inibição indicam pontos de similaridades e diferenças entre N. caninum, Plasmodium e T. gondii. Por exemplo, a atovaquona demonstra uma alta atividade antiparasitária em todos os modelos testados, enquanto a cloroquina não inibe N. caninum. Por outro lado, a tetraciclina é efetiva contra Plasmodium e N. caninum, em contraste com a baixa atividade em modelos de T. gondii. O reposicionamento de drogas antimaláricas em N. caninum é uma forma rápida e de baixo custo para o desenvolvimento de novas formulações que usam compostos bem estabelecidos.(AU)

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A meningite bacteriana é uma importante causa de morbidade e mortalidade, sendo mundialmente reconhecida como problema de saúde pública. Em neonatos, a ocorrência da doença logo nas primeiras semanas de vida sugere a possibilidade de transmissão vertical, observada em muitos casos de infecção pelo Streptococcus agalactiae. O S. agalactiae é considerado um dos principais causadores de meningite neonatal, sendo esta a manifestação clínica de 16% das infecções de início precoce e de 43% das de início tardio. São descritos diversos fatores de virulência que contribuem para a patogenicidade da bactéria e o desenvolvimento da meningite, sendo os neonatos muito suscetíveis à doença invasiva em decorrência das deficiências qualitativas e quantitativas relacionadas à fagocitose, anticorpos específicos e vias clássica e alternativa do complemento. Com isso, este trabalho teve como objetivo a avaliação da ocorrência dos casos de meningite por S. agalactiae abordando seus aspectos microbiológicos e condutas de prevenção, a fim de ressaltar a importância deste agente na vigilância das meningites...(AU)

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Neospora caninum is a member of Apicomplexa phylum, the causative agent of neosporosis. The neosporosis combat is not well established and several strategies related to vaccine, chemotherapy and immune modulation are under development. In this work, we evaluated the effects of artemisinin (Art), methylene blue (MB) and pyrimethamine (Pyr) alone or associated, on N. caninum proliferation and elimination using LacZ tagged tachyzoites. The reactive oxygen species (ROS) production after incubation with Art were also performed. Our results indicate that combinations of classical antimalarial drugs improve the parasite control, allowing the use of three drugs in a single dose. Additionally, artemisinin demonstrated distinct ROS production patterns in intra and extracellular N. caninum forms. The drug repurposing appears as a suitable approach, allowing a fast and safe method to evaluate old drugs but novel candidates against neosporosis.

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