Multidrug-resistant Escherichia coli strains isolated from swine manure biofertilizer in Brazil.

Escherichia coli is one of the key bacteria responsible for a variety of diseases in humans and livestock-associated infections around the globe. It is the leading cause of mortality in neonatal and weaned piglets in pig husbandry, causing diarrhea and significant harm to the industry. Furthermore, the frequent and intensive use of antimicrobials for the prevention of diseases, particularly gastrointestinal diseases, may promote the selection of multidrug-resistant (MDR) strains. These resistant genotypes can be transmitted through the excrement of animals, including swine. It is common practice to use porcine manure processed by biodigesters as fertilizer. This study aimed to examine the antimicrobial susceptibility, the presence of virulence genes frequently associated with pathotypes of intestinal pathogenic E. coli (InPEC), and antimicrobial resistance genes (ARGs) of 28 E. coli isolates collected from swine manure fertilizers. In addition, the enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) technique was used to investigate the genetic relationship among the strains. Using disk diffusion, the antimicrobial susceptibility profiles of the strains were determined. Using polymerase chain reaction (PCR), 14 distinct virulence genes associated with the most prevalent diarrhea and intestinal pathogenic E. coli (DEC/InPEC) and five ARGs were analyzed. All isolates tested positive for multidrug resistance. There was no detection of any of the 14 virulence genes associated with InPECs, indicating the presence of an avirulent commensal microbiota. Molecular classification by ERIC-PCR revealed that the majority of isolates (27 isolates) coalesced into a larger cluster with a genetic similarity of 47.7%; only one strain did not cluster in this cluster, indicating a high level of genetic diversity among the analyzed isolates. Thus, it is of the utmost importance to conduct epidemiological surveillance of animal breeding facilities in order to determine their microbiota and formulate plans to reduce the use of antimicrobials and improve animal welfare.

Long-term occurrence of multiple antimicrobial drug resistant Klebsiella pneumoniae isolates harboring virulent potential in a tertiary hospital from Brazil.

Klebsiella pneumoniae strains are globally associated with a plethora of opportunistic and severe human infections and are known to spread genes conferring antimicrobial resistance. Some strains harbor virulence determinants that enable them to cause serious disease in any patient, both in the hospital and in the community. The aim of this study was to determine the frequency of antimicrobial resistance and virulence traits (by gene detection and string test) among 83 K. pneumoniae isolates obtained from patient cultures of a scholar tertiary hospital in the Midwestern Brazil (Brasília, DF). Antimicrobial susceptibility analysis showed that 94% (78/83) of the isolates presented one of the following resistance profiles: resistant (R, 39), multidrug-resistant (MDR, 29), or extensively drug-resistant (XDR, 10). Several MDR and XDR strains harbored multiple virulence genes and displayed hypermucoviscous phenotype. These characteristics were observed among isolates obtained throughout all the sample collection period (2013 - 2017). The K2 serotype gene, a molecular marker of hypervirulence, was detected in three isolates, one of which classified as XDR. Sequence typing revealed the occurrence of isolates belonged to high-risk (ST13) and multiple resistance-spreading clones (ST105). Thus, our findings showed the occurrence of virulent potential isolates that also presented MDR/XDR phenotypes from 2013 to 2015. This study also indicates the probable convergence of virulence and resistance since at least 2013 in Brazil.

Identification and antimicrobial susceptibility profile of bacteria isolated from primary endodontic infections.

This study aimed to identify and characterize the antimicrobial susceptibility profile of bacteria found in primary endodontic infections in the teeth of patients treated at the Dental Clinic of the University of Ribeirão Preto, São Paulo, Brazil. From September to December 2019, samples were obtained from 21 patients with primary endodontic infections. The collections were carried out in triplicate using paper cones placed close to the total length of the root canal. Bacterial isolation was performed in Brain Heart Infusion agar, Blood agar, and other selective culture media cultured at 37°C for up to 48 h under aerobiosis and microaerophilic conditions. The bacterial species were identified using the Vitek 2 automated system. The disk diffusion method on agar Müeller-Hinton was used to assess antimicrobial susceptibility with the recommended antimicrobials for each identified bacterial species. A total of 49 antibiotics were evaluated. Fifteen of the 21 samples collected showed bacterial growth, and 17 bacterial isolates were found. There were 10 different bacterial species identified: Enterococcus faecalis (four isolates), Streptococcus mitis/oralis (three isolates), Streptococcus anginosus (three isolates) being the most common, followed by Staphylococcus epidermidis, Enterococcus faecium, Streptococcus constellatus, Streptococcus alactolyticus, Enterobacter cloacae, Klebsiella variicola, and Providencia rettgeri (one isolate of each species). The analysis demonstrated significant susceptibility to most of the tested antibiotics. However, some Enterococcus isolates resisted the antibiotic's erythromycin, ciprofloxacin, and tetracycline. A Staphylococcus epidermidis isolate was characterized as multidrug-resistant. Five Streptococcus isolates were non-susceptible to all antibiotics tested.

Identification and antimicrobial susceptibility profile of bacteria isolated from primary endodontic infections

Abstract This study aimed to identify and characterize the antimicrobial susceptibility profile of bacteria found in primary endodontic infections in the teeth of patients treated at the Dental Clinic of the University of Ribeirão Preto, São Paulo, Brazil. From September to December 2019, samples were obtained from 21 patients with primary endodontic infections. The collections were carried out in triplicate using paper cones placed close to the total length of the root canal. Bacterial isolation was performed in Brain Heart Infusion agar, Blood agar, and other selective culture media cultured at 37°C for up to 48 h under aerobiosis and microaerophilic conditions. The bacterial species were identified using the Vitek 2 automated system. The disk diffusion method on agar Müeller-Hinton was used to assess antimicrobial susceptibility with the recommended antimicrobials for each identified bacterial species. A total of 49 antibiotics were evaluated. Fifteen of the 21 samples collected showed bacterial growth, and 17 bacterial isolates were found. There were 10 different bacterial species identified: Enterococcus faecalis (four isolates), Streptococcus mitis/oralis (three isolates), Streptococcus anginosus (three isolates) being the most common, followed by Staphylococcus epidermidis, Enterococcus faecium, Streptococcus constellatus, Streptococcus alactolyticus, Enterobacter cloacae, Klebsiella variicola, and Providencia rettgeri (one isolate of each species). The analysis demonstrated significant susceptibility to most of the tested antibiotics. However, some Enterococcus isolates resisted the antibiotic's erythromycin, ciprofloxacin, and tetracycline. A Staphylococcus epidermidis isolate was characterized as multidrug-resistant. Five Streptococcus isolates were non-susceptible to all antibiotics tested.

Multidrug-resistant Klebsiella pneumoniae: a retrospective study in Manaus, Brazil.

Klebsiella pneumoniae is an opportunistic pathogen that can cause several infections, mainly in hospitalised or immunocompromised individuals. The spread of K. pneumoniae emerging virulent and multidrug-resistant clones is a worldwide concern and its identification is crucial to control these strains especially in hospitals. This article reports data related to multi-resistant K. pneumoniae strains, isolated from inpatients in the city of Manaus, Brazil, harbouring virulence and antimicrobial-resistance genes, including high-risk international clones belonging to clonal group (CG) 258. Twenty-one strains isolated from different patients admitted to four hospitals in the city of Manaus, located in the state of Amazonas, Northern Brazil (Amazon Rainforest region) were evaluated. The majority of strains (61.9% n = 13) were classified as multidrug-resistant (MDR), and five strains (23.8%) as extensively drug-resistant (XDR). Several virulence and antimicrobial-resistance genes were found among the strains and eight strains (38.1%) presented the hyper-mucoviscous phenotype. MLST analysis demonstrated a great diversity of STs among the strains, totaling 12 different STs (ST11, ST23, ST198, ST277, ST307, ST340, ST378, ST462, ST502, ST3991, ST3993 and ST5209). Three of these (ST11, ST23 and ST340) belong to CG258.

A novel hypermucoviscous Klebsiella pneumoniae ST3994-K2 clone belonging to Clonal Group 86.

Emergent hypervirulent Klebsiella pneumoniae has been responsible for severe diseases, representing a serious threat to public health. We report the whole-genome sequencing of a novel ST3994-K2 clone, a single locus variant of ST86 K2, which is considered a worrying hypervirulent clone that emerged in several parts of the world. The strain K. pneumonia Kpi144 was isolated in 2013 from a blood culture of a 69-year-old male patient admitted to a tertiary hospital in Teresina, state of Piauí, northeastern Brazil. The strain was susceptible to 41 antibiotics tested, presented hypermucoviscous phenotype and a virulent behavior was observed in the Galleria mellonella infection model. Moreover, the virulome showed several virulence genes. To the best of our knowledge, this is the first worldwide report of a novel ST3994-K2 K. pneumoniae clone, an SLV of ST86 K2, which is considered a worrying virulent clone that has emerged in several parts of the world, including South America and Brazil.

The primary motor cortex electrical and chemical stimulation attenuates the chronic neuropathic pain by activation of the periaqueductal grey matter: The role of NMDA receptors.

BACKGROUND: Motor cortex stimulation (MCS) is proper as a non-pharmacological therapy for patients with chronic and neuropathic pain (NP). AIMS: This work aims to investigate if the MCS in the primary motor cortex (M1) produces analgesia and how the MCS could interfere in the MCS-induced analgesia. Also, to elucidate if the persistent activation of N-methyl-d-aspartic acid receptor (NMDAr) in the periaqueductal grey matter (PAG) can contribute to central sensitisation of the NP. METHODS: Male Wistar rats were submitted to the von Frey test to evaluate the mechanical allodynia after 21 days of chronic constriction injury (CCI) of the sciatic nerve. The MCS was performed with low-frequency (20 µA, 100 Hz) currents during 15 s by a deep brain stimulation (DBS) device. Moreover, the effect of M1-treatment with an NMDAr agonist (at 2, 4, and 8 nmol) was investigated in CCI rats. The PAG dorsomedial column (dmPAG) was pretreated with the NMDAr antagonist LY 235959 (at 8 nmol), followed by MCS. RESULTS: The MCS decreased the mechanical allodynia in rats with chronic NP. The M1-treatment with an NMDA agonist at 2 and 8 nmol reduced the mechanical allodynia in CCI rats. In addition, dmPAG-pretreatment with LY 235959 at 8 nmol attenuated the mechanical allodynia evoked by MCS. CONCLUSION: The M1 cortex glutamatergic system is involved in the modulation of chronic NP. The analgesic effect of MCS may depend on glutamate signaling recruitting NMDAr located on PAG neurons in rodents with chronic NP.

Characterization of multidrug-resistant and virulent Klebsiella pneumoniae strains belonging to the high-risk clonal group 258 (CG258) isolated from inpatients in northeastern Brazil.

Multidrug-resistant (MDR) and hypervirulent Klebsiella pneumoniae (hvKp) clones have become a major threat to global public health. The clonal group 258 (CG258) is considered a high-risk CG and the K. pneumoniae strains belonging to it are often multi-resistant and to spread mainly in the hospital environment. This study aimed to characterize the antimicrobial resistance profile, virulence factors, and the clonal relationships among 13 K. pneumoniae strains belonging to CG258 from patients admitted to a tertiary hospital in Teresina, in the state of Piauí, northeastern Brazil. Ten strains were classified as MDR and three as extensively drug-resistant (XDR). Three different ß-lactamase-encoding genes (blaKPC, blaOXA-1-like, and blaCTX-M-Gp1) and six virulence genes (fimH, ycfM, mrkD, entB, ybtS, and kfu) were detected. Moreover, two hypermucoviscous K. pneumoniae strains and one capsular K-type 2 were found. Multilocus sequence typing analysis revealed ten different sequence types (STs) (ST14, ST17, ST20, ST29, ST45, ST101, ST268, ST1800, ST3995, and ST3996) belonging to CG258, being two (ST3995 and ST3996) described for the first time in this study.

Multidrug-Resistant (MDR) Klebsiella variicola Strains Isolated in a Brazilian Hospital Belong to New Clones.

Klebsiella variicola is mainly associated with opportunistic infections and frequently identified as Klebsiella pneumoniae. This misidentification implies a wrong epidemiology result as well as incorrect attribution to K. pneumoniae as the etiology of some severe infections. Recently, huge efforts have been made to study K. variicola, however, the biological aspects of this species are still unclear. Here we characterized five K. variicola strains initially identified as K. pneumoniae, with a Vitek-2 System and 16S rRNA sequencing. One-step multiplex polymerase chain reaction and Whole Genome Sequencing (WGS) identified them as K. variicola. Additionally, WGS analysis showed that all the strains are closely related with K. variicola genomes, forming a clustered group, apart from K. pneumoniae and K. quasipneumoniae. Multilocus sequence typing analysis showed four different sequence types (STs) among the strains and for two of them (Kv97 and Kv104) the same ST was assigned. All strains were multidrug-resistant (MDR) and three showed virulence phenotypes including invasion capacity to epithelial cells, and survival in human blood and serum. These results showed the emergence of new K. variicola clones with pathogenic potential to colonize and cause infection in different tissues. These characteristics associated with MDR strains raise great concern for human health.

Antibacterial Activity of a New Ready-To-Use Calcium Silicate-Based Sealer.

The aim of this study was to evaluate the antibacterial potential of a calcium silicate-based sealer (Bio-C Sealer, Angelus) against common bacteria in primary and secondary endodontic infections. Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus mutans were exposed to fresh Bio-C Sealer for 24 h by the agar diffusion method (n=5). Additionally, the antibacterial activity was investigated against E. faecalis and S. mutans biofilms (48 h old) grown in discs with 4 mm in diameter and 2 mm in height. (n=3) of set discs of Bio-C Sealer (Angelus), EndoFill (Dentsply-Mallefer), Sealer 26 (Dentsply), AH Plus (Dentsply), Sealapex (Sybron-Endo) and EndoSequence BC Sealer (Brasseler). The antibacterial activity was evaluated by colony forming unity (CFU) counting using ImageJ software. Data were compared by one-way ANOVA followed by Holm-Sidak test (a=5%). Fresh Bio-C Sealer exhibited antimicrobial activity against all bacteria evaluated by agar diffusion method, except for S. mutans. Set discs of all endodontic sealers tested showed similar CFU values for E. faecalis (p>0.05). S. mutans in biofilms showed higher susceptibility to EndoFill compared with the other sealers (p<0.05). In conclusion, the results indicate that fresh Bio-C Sealer does not inhibit S. mutans growth, but exhibits antibacterial activity against E. faecalis, S. aureus, P. aeruginosa and E. coli. After setting, the Bio-C Sealer exhibits an antimicrobial potential comparable to that of the other sealers evaluated in E. faecalis biofilm, but lower than that of EndoFill for S. mutans biofilm.

Antibacterial Activity of a New Ready-To-Use Calcium Silicate-Based Sealer

Abstract The aim of this study was to evaluate the antibacterial potential of a calcium silicate-based sealer (Bio-C Sealer, Angelus) against common bacteria in primary and secondary endodontic infections. Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus mutans were exposed to fresh Bio-C Sealer for 24 h by the agar diffusion method (n=5). Additionally, the antibacterial activity was investigated against E. faecalis and S. mutans biofilms (48 h old) grown in discs with 4 mm in diameter and 2 mm in height. (n=3) of set discs of Bio-C Sealer (Angelus), EndoFill (Dentsply-Mallefer), Sealer 26 (Dentsply), AH Plus (Dentsply), Sealapex (Sybron-Endo) and EndoSequence BC Sealer (Brasseler). The antibacterial activity was evaluated by colony forming unity (CFU) counting using ImageJ software. Data were compared by one-way ANOVA followed by Holm-Sidak test (a=5%). Fresh Bio-C Sealer exhibited antimicrobial activity against all bacteria evaluated by agar diffusion method, except for S. mutans. Set discs of all endodontic sealers tested showed similar CFU values for E. faecalis (p>0.05). S. mutans in biofilms showed higher susceptibility to EndoFill compared with the other sealers (p<0.05). In conclusion, the results indicate that fresh Bio-C Sealer does not inhibit S. mutans growth, but exhibits antibacterial activity against E. faecalis, S. aureus, P. aeruginosa and E. coli. After setting, the Bio-C Sealer exhibits an antimicrobial potential comparable to that of the other sealers evaluated in E. faecalis biofilm, but lower than that of EndoFill for S. mutans biofilm.

Resumo O objetivo deste estudo foi avaliar o potencial antibacteriano do novo cimento biocerâmico (Bio-C Sealer, Angelus) contra bactérias comuns em infecções endodônticas primárias e secundárias. Culturas de Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus e Streptococcus mutans foram expostos a amostras frescas do Bio-C sealer durante 24 h pelo método de difusão em agar (n=5). A atividade antibacteriana de amostras dos cimentos Bio-C Sealer (Angelus), EndoFill (Dentsply-Mallefer), Sealer 26 (Dentsply), AH Plus (Dentsply), Sealapex (Sybron-Endo) e EndoSequence BC Sealer (Brasseler) após a presa também foi investigada em biofilmes de 48 h das bactérias E. faecalis e S. mutans, crescidos em discos com 4 mm de diâmetro e 2 mm de altura. A atividade antibacteriana foi avaliada por contagem das unidades formadoras de colônias (UFC) utilizando o software ImageJ. Os dados foram comparados por ANOVA a um critério seguido pelo pós-teste Holm-Sidak (a=5%). Amostras frescas do Bio-C Sealer exibiram atividade antimicrobiana contra todas as bactérias avaliadas pelo método de difusão em ágar, exceto para S. mutans. A análise da formação de biofilme mostrou que todos os cimentos endodônticos testados apresentaram valores similares de UFC para E. faecalis (p> 0,05), enquanto biofilmes de S. mutans foram mais suscetíveis ao EndoFill em comparação com os demais cimentos (p<0,05). Conclui-se que o cimento Bio-C Sealer fresco exibe atividade antibacteriana para E. faecalis, S. aureus, P. aeruginosa e E. coli, mas não inibe o crescimento de S. mutans. Após a presa, o cimento Bio-C Sealer exibe potencial antimicrobiano similar ao dos demais cimentos avaliados em biofilme de E. faecalis, mas inferior ao do EndoFill para S. mutans.

CB1-cannabinoid-, TRPV1-vanilloid- and NMDA-glutamatergic-receptor-signalling systems interact in the prelimbic cerebral cortex to control neuropathic pain symptoms.

Neuropathic pain (NP) is a challenge due to our limited understanding of the mechanisms that initiate and maintain chronic pain. The prelimbic division (PrL) of the medial prefrontal cortex (mPFC) is an important area of the emotional and cognitive components of pain and pharmacological systems can interact into the neocortex to elaborate the chronic pain. This work aimed to investigate the pharmacological cross-talk between synaptic neurotransmission, neuroanatomical approaches and NP conditions. A bidirectional neural tract tracer, the 3000-molecular-weight biodextran (BDA) was microinjected into the PrL cortex. The mechanical withdrawal threshold (MWT) was recorded by a von Frey test, and the effect of prelimbic cortex CB1, NMDA, and TRPV1 receptor modulation was evaluated 21 days after chronic constriction injury (CCI) of the sciatic nerve in male Wistar rats. Microinjection of a bidirectional neurotracer in the PrL cortex showed connections with the lateral division of the mediodorsal thalamic nucleus (MDL), central division of the mediodorsal thalamic nucleus (MDC), centrolateral thalamic nucleus (CL), ventromedial thalamic nucleus (VM), and the paracentral thalamic nucleus (PC). In detail, AM251, a CB1 receptor antagonist (at 50, 100 and 200 pmol) microinjections intra-PrL cortex decreased the MWT. Administrations of 6-iodonordihydrocapsaicin (6-I-CPS), a transient receptor potential vanilloid type 1 (TRPV1) antagonist, at 3 nmol and the endocannabinoid anandamide (AEA) at 50 and 100 pmol increased the MWT. AEA at 200 pmol injected in the PrL cortex decreased the MWT, and this hyperalgesic effect was blocked by 6-I-CPS at 3 nmol. The AEA (at 100 pmol) anti-allodynic effect was attenuated by AM251 (at 5 pmol). The TRPV1 selective agonist N-oleoyldopamine (OLDA) at 10 µM decreased the MWT. The blockade of the NMDA receptor with LY235959 (at 8 nmol) and 6-I-CPS (at 3 nmol) reversed the OLDA (at 10 µM) hyperalgesic effect. These findings showed that the PrL cortex sends pathways to thalamic nuclei that can mediate the nociception. We also suggest that the PrL cortex is involved in the potentiation and maintenance of mechanical allodynia by NMDA and TRPV1 receptor activation and that attenuation of this allodynia depends on CB1 receptor activation during NP.

Influence of different ceramic materials and surface treatments on the adhesion of Prevotella intermedia.

Ceramics are used in oral rehabilitation; however, these materials are prone to formation of biofilms that may cause periodontal diseases. This study aimed to evaluate the influence of distinct surface treatments on ceramic surface roughness and biofilm formation of oral bacteria (Prevotella intermedia). Eighty-four specimens of the following four ceramic systems were produced: LC - leucite-based glass ceramic, LD - lithium disilicate-based glass ceramic, LSZ - glass ceramic based on zirconia-reinforced lithium silicate, and ZR - monolithic zirconia. These were submitted to three different surface treatment protocols: C - control, G - glazing, and GDB - grinding with diamond bur (n = 7). The surface characteristics were assessed using a confocal laser microscope (Ra) and a scanning electron microscope (SEM). Thereafter, the groups were contaminated with a bacterial strain of P. intermedia ATCC 25611. The biofilms formed were quantified by counting the colony forming units (CFUs) and analyzed with scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM). Data were analyzed by using a 2-way ANOVA and the Tukey test (ɑ = 0.05). Results showed that greater roughness was associated with GDB (p < 0.05). The same was also true for the ceramic material ZR (p < 0.05). There was a statistical significant difference in the CFU counts between the materials (p < 0.05) that revealed a greater amount of bacterial adhesion in the LC and ZR groups (p > 0.05). Thus, it was suggested that the surface roughness of the ceramic materials favored bacterial adhesion; and thus, finishing of ceramic surfaces with GDB should be avoided.

Characterization of KPC-Producing Serratia marcescens in an Intensive Care Unit of a Brazilian Tertiary Hospital.

Serratia marcescens has emerged as an important opportunistic pathogen responsible for nosocomial and severe infections. Here, we determined phenotypic and molecular characteristics of 54 S. marcescens isolates obtained from patient samples from intensive-care-unit (ICU) and neonatal intensive-care-unit (NIUC) of a Brazilian tertiary hospital. All isolates were resistant to beta-lactam group antibiotics, and 92.6% (50/54) were not susceptible to tigecycline. Furthermore, 96.3% showed intrinsic resistance to polymyxin E (colistin), a last-resort antibiotic for the treatment of infections caused by MDR (multidrug-resistant) Gram-negative bacteria. In contrast, high susceptibility to other antibiotics such as fluoroquinolones (81.5%), and to aminoglycosides (as gentamicin 81.5%, and amikacin 85.2%) was found. Of all isolates, 24.1% were classified as MDR. The presence of resistance and virulence genes were examined by PCR and sequencing. All isolates carried KPC-carbapenemase (bla KPC ) and extended spectrum beta-lactamase bla TEM genes, 14.8% carried bla OXA- 1, and 16.7% carried bla CTX-M- 1 group genes, suggesting that bacterial resistance to ß-lactam antibiotics found may be associated with these genes. The genes SdeB/HasF and SdeY/HasF that are associated with efflux pump mediated drug extrusion to fluoroquinolones and tigecycline, respectively, were found in 88.9%. The aac(6')-Ib-cr variant gene that can simultaneously induce resistance to aminoglycoside and fluoroquinolone was present in 24.1% of the isolates. Notably, the virulence genes to (i) pore-forming toxin (ShlA); (ii) phospholipase with hemolytic and cytolytic activities (PhlA); (iii) flagellar transcriptional regulator (FlhD); and (iv) positive regulator of prodigiosin and serratamolide production (PigP) were present in 98.2%. The genetic relationship among the isolates determined by ERIC-PCR demonstrated that the vast majority of isolates were grouped in a single cluster with 86.4% genetic similarity. In addition, many isolates showed 100% genetic similarity to each other, suggesting that the S. marcescens that circulate in this ICU are closely related. Our results suggest that the antimicrobial resistance to many drugs currently used to treat ICU and NIUC patients, associated with the high frequency of resistance and virulence genes is a worrisome phenomenon. Our findings emphasize the importance of active surveillance plans for infection control and to prevent dissemination of these strains.

In Silico Identification of New Targets for Diagnosis, Vaccine, and Drug Candidates against Trypanosoma cruzi.

Chagas disease is a neglected tropical disease caused by the parasite Trypanosoma cruzi. Despite the efforts and distinct methodologies, the search of antigens for diagnosis, vaccine, and drug targets for the disease is still needed. The present study is aimed at identifying possible antigens that could be used for diagnosis, vaccine, and drugs targets against T. cruzi using reverse vaccinology and molecular docking. The genomes of 28 T. cruzi strains available in GenBank (NCBI) were used to obtain the genomic core. Then, subtractive genomics was carried out to identify nonhomologous genes to the host in the core. A total of 2630 conserved proteins in 28 strains of T. cruzi were predicted using OrthoFinder and Diamond software, in which 515 showed no homology to the human host. These proteins were evaluated for their subcellular localization, from which 214 are cytoplasmic and 117 are secreted or present in the plasma membrane. To identify the antigens for diagnosis and vaccine targets, we used the VaxiJen software, and 14 nonhomologous proteins were selected showing high binding efficiency with MHC I and MHC II with potential for in vitro and in vivo tests. When these 14 nonhomologous molecules were compared against other trypanosomatids, it was found that the retrotransposon hot spot (RHS) protein is specific only for T. cruzi parasite suggesting that it could be used for Chagas diagnosis. Such 14 proteins were analyzed using the IEDB software to predict their epitopes in both B and T lymphocytes. Furthermore, molecular docking analysis was performed using the software MHOLline. As a result, we identified 6 possible T. cruzi drug targets that could interact with 4 compounds already known as antiparasitic activities. These 14 protein targets, along with 6 potential drug candidates, can be further validated in future studies, in vivo, regarding Chagas disease.

Nanoparticle-Mediated Drug Delivery: Blood-Brain Barrier as the Main Obstacle to Treating Infectious Diseases in CNS.

BACKGROUND: Parasitic infections affecting the central nervous system (CNS) present high morbidity and mortality rates and affect millions of people worldwide. The most important parasites affecting the CNS are protozoans (Plasmodium sp., Toxoplasma gondii, Trypanosoma brucei), cestodes (Taenia solium) and free-living amoebae (Acantamoeba spp., Balamuthia mandrillaris and Naegleria fowleri). Current therapeutic regimens include the use of traditional chemicals or natural compounds that have very limited access to the CNS, despite their elevated toxicity to the host. Improvements are needed in drug administration and formulations to treat these infections and to allow the drug to cross the blood-brain barrier (BBB). METHODS: This work aims to elucidate the recent advancements in the use of nanoparticles as nanoscaled drug delivery systems (NDDS) for treating and controlling the parasitic infections that affect the CNS, addressing not only the nature and composition of the polymer chosen, but also the mechanisms by which these nanoparticles may cross the BBB and reach the infected tissue. RESULTS: There is a strong evidence in the literature demonstrating the potential usefulness of polymeric nanoparticles as functional carriers of drugs to the CNS. Some of them demonstrated the mechanisms by which drugloaded nanoparticles access the CNS and control the infection by using in vivo models, while others only describe the pharmacological ability of these particles to be utilized in in vitro environments. CONCLUSION: The scarcity of the studies trying to elucidate the compatibility as well as the exact mechanisms by which NDDS might be entering the CNS infected by parasites reveals new possibilities for further exploratory projects. There is an urgent need for new investments and motivations for applying nanotechnology to control parasitic infectious diseases worldwide.

New sequence type in multidrug-resistant Klebsiella pneumoniae harboring the blaNDM-1-encoding gene in Brazil.

OBJECTIVES: The aim of this study was to investigate the blaNDM gene, pathogenic potential, and antimicrobial resistance of clinical isolates of carbapenem-resistant Klebsiella pneumoniae isolated from patients admitted to the University Hospital of Londrina between January 2014 and March 2017. METHODS: blaNDM-1 and virulence genes were investigated using conventional PCR methods Antimicrobial susceptibility testing was performed by disk diffusion method according to the Clinical and Laboratory Standards Institute guidelines of 2017. Clonal relationships of the New Delhi metallo-ß-lactamase (NDM)-positive isolates were determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus sequence typing (MLST). RESULTS: A total of 825 K. pneumoniae were identified, with four isolates (Kp6408, Kp6410, Kp6411, and Kp6715) presenting the blaNDM-1 gene. All NDM-1-producing isolates showed co-production of blaKPC-2 and blaTEM genes and also the virulence genes kfu, entB, mrkD, and fimH. Three isolates (Kp6408, Kp6410, and Kp6715) were classified as multidrug-resistant (MDR) and one (Kp6411) as extensively drug-resistant (XDR). ERIC-PCR analyses demonstrated that the isolates shared about 60% genetic similarity. MLST revealed four different sequence types (STs), described for the first time in Brazil, with two novel STs described in this study: ST3371 and ST3372. CONCLUSION: This study reports the identification of NDM-1 associated with KPC and virulence genes in four MDR K. pneumoniae with STs first described in Brazil.

Detection of virulence and ß-lactamase encoding genes in Enterobacter aerogenes and Enterobacter cloacae clinical isolates from Brazil.

Enterobacter cloacae and E. aerogenes have been increasingly reported as important opportunistic pathogens. In this study, a high prevalence of multi-drug resistant isolates from Brazil, harboring several ß-lactamase encoding genes was found. Several virulence genes were observed in E. aerogenes, contrasting with the E. cloacae isolates which presented none.