Gut microbiota modulation by prednisolone in a rat kindling model of pentylenetetrazol (PTZ)-induced seizure.

The gut microbiota is a complex community composed by several microorganisms that interact in the maintenance of homeostasis and contribute to physiological processes, including brain function. The relationship of the taxonomic composition of the gut microbiota with neurological diseases such as autism, Parkinson's, Alzheimer's, anxiety, and depression is widely recognized. The immune system is an important intermediary between the gut microbiota and the central nervous system, being one of the communication routes of the gut-brain axis. Although the complexity of the relationship between inflammation and epilepsy has not yet been elucidated, inflammatory processes are similar in many ways to the consequences of dysbiosis and contribute to disease progression. This study aimed to analyze the taxonomic composition of the gut microbiota of rats treated with prednisolone in a kindling model of epilepsy. Male Wistar rats (90 days, n = 24) divided into four experimental groups: sodium chloride solution 0.9 g%, diazepam 2 mg/kg, prednisolone 1 mg/kg, and prednisolone 5 mg/kg administered intraperitoneally (i.p.) for 14 days. The kindling model was induced by pentylenetetrazole (PTZ) 25 mg/kg i.p. on alternate days. The taxonomic profile was established by applying metagenomic DNA sequencing. There was no change in alpha diversity, and the composition of the gut microbiota between prednisolone and diazepam was similar. The significant increase in Verrucomicrobia, Saccharibacteria, and Actinobacteria may be related to the protective activity against seizures and inflammatory processes that cause some cases of epilepsy. Further studies are needed to investigate the functional influence that these species have on epilepsy and the inflammatory processes that trigger it.

High rate of Clostridioides difficile colonization in patients admitted to intensive care: A prospective cohort study.

Here, we evaluated the frequency of C. difficile colonization and its impact on clinical outcomes in patients admitted to intensive care units in Brazil. From ninety-two patients screened 16 (17.3%) were colonized by C. difficile. Colonized patients had higher Simplified Acute Physiology Score III (SAPS III), however there was no association between C. difficile colonization with diarrhea or mortality. The C. difficile strains sequenced belonged to clade 1 and presented high vancomycin-resistant rates.

Pooling of samples to optimize SARS-CoV-2 diagnosis by RT-qPCR: comparative analysis of two protocols.

This study has aimed to evaluate the use pool of samples as a strategy to optimize the diagnostic of SARS-CoV-2 by RT-qPCR. A total of 220 naso/orofaryngeal swab samples were collected and tested using two different protocols of sample pooling. Results from protocol A were identical with the individual results. However, for results from protocol B, reduced agreement (91%) was observed in relation to individual testing. Inconsistencies observed were related to RT-qPCR results with higher cycle thresholds. These results suggest that pooling of samples before RNA extraction is preferable in terms of diagnostic for SARS-CoV-2.

Hepatoprotective Effect of Probiotic Lactobacillus rhamnosus GG Through the Modulation of Gut Permeability and Inflammasomes in a Model of Alcoholic Liver Disease in Zebrafish.

Objective: Alcoholic liver disease (ALD) is among the leading causes of death from liver disease. Among the factors involved in its pathogenesis are inflammation and increased intestinal permeability. The aim of this study was to assess the effect of Lactobacillus rhamnosus GG (LGG) on hepatic lipid accumulation, activation of inflammasomes, and gut permeability markers in experimental model of ALD with zebrafish.Methods: An experiment was conducted to assess the effective LGG dose capable of promoting intestinal colonization. Animals were divided into three groups (n = 64/group): ethanol group (E), ethanol + probiotic group (EP), and control group (C). Groups E and EP were exposed to 0.5% ethanol concentration for 28 days. At the end of this period, animals were euthanized, and livers were collected for Oil Red staining and assessment of the inflammasome system. Intestines were collected for evaluation of gut permeability markers.Results: The dose of 1.55 × 106 UFC LGG/fish/d promoted intestinal colonization. Group EP presented lower hepatic lipid accumulation, lower il-1ß expression, and higher cldn15a expression when compared to group E.Conclusions: Supplementation with LGG was protective for hepatic steatosis in ALD model. In addition, LGG influenced the modulation of the inflammatory response and markers of gut permeability, improving the gut barrier structure.

Salmonella enterica mcr-1 Positive from Food in Brazil: Detection and Characterization.

The mcr-1 gene has been identified in bacterial isolates obtained from humans, animals, environment, and food, including Salmonella spp., which is one of the major foodborne pathogens worldwide. The aim of this study was to evaluate the presence of mcr-1 gene in Salmonella spp. from food produced in Brazil and to characterize the isolates harboring this gene. A total of 490 Salmonella spp. isolates from the Brazilian National Program for the Control of Foodborne Pathogens were screened for the presence of mcr-1 gene by polymerase chain reaction (PCR). Whole genome sequencing (WGS) was performed in positive isolates to characterize the sequence type (ST), plasmid families and resistance genes. Antimicrobial susceptibility tests were performed by broth microdilution. Selected isolates were submitted to conjugation experiments using the Escherichia coli J53 as a receptor. We detected eight isolates harboring the mcr-1 gene; seven belonged to Salmonella enterica serovar Typhimurium and its monophasic variant 4,[5],12:i:-, and one belonged to serovar Saintpaul. Seven of the mcr-1 positive isolates displayed a high rate of resistance to other antibiotics in addition to colistin. Analysis of the WGS indicated that the ST 19 was the most common ST among the mcr-1 positive isolates. The mcr-1 gene was located in an IncX4 plasmid of ∼33 kb, with no additional resistance genes and with high identity with a plasmid obtained from a clinical isolate of E. coli mcr-1 positive in Brazil. All plasmids harboring the mcr-1 gene were able to conjugate. Our results suggest the spread of a single plasmid type in Brazil harboring the mcr-1 among Salmonella spp. The horizontal transfer of this mobile element has been contributing to the spread of the colistin resistance in the country.

Rapid Detection of Carbapenemase Production Directly from Blood Culture by Colorimetric Methods: Evaluation in a Routine Microbiology Laboratory.

The aim of this study was to evaluate the two rapid colorimetric methods (CNPt-Direct and Blue-Carba) for the detection of carbapenemase production directly from blood culture in a routine microbiology laboratory. The methods were initially evaluated on spiked blood cultures with 61 carbapenemase-positive isolates. Afterwards, they were used in blood cultures (314 samples were evaluated) obtained from patients in a routine microbiology laboratory during a period of 6 months. The colorimetric methods were compared to the conventional culture of blood. The results of the spiked blood cultures indicated that both colorimetric methods presented positive results for the vast majority (95%) of the isolates harboring KPC, NDM, and IMP genes. However, the assay failed to detect many GES- and OXA-48-like-positive isolates (65% positive results). In the second part of the study, a total of 314 blood cultures from patients were evaluated, and 33 yielded Enterobacteriaceae isolates resistant to meropenem (30 isolates were positive for carbapenemases according to PCR). The colorimetric tests correctly detected 24 out of the 30 carbapenemase-positive isolates directly from the blood vial (80% positive results). Overall positive percent agreement and negative percent agreement were 80% and 100%, respectively. The colorimetric assays are simple and cost-effective methods that can be implemented in a routine microbiology laboratory, diminishing the time necessary to detect carbapenemase-producing isolates from 24 to 48 h to 3 to 5 h. Moreover, according to our results, the positive colorimetric test results do not need to be confirmed and can be immediately provided to the attending physician.

Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR.

Linking Microbial Community and Catabolic Gene Structures during the Adaptation of Three Contaminated Soils under Continuous Long-Term Pollutant Stress.

Three types of contaminated soil from three geographically different areas were subjected to a constant supply of benzene or benzene/toluene/ethylbenzene/xylenes (BTEX) for a period of 3 months. Different from the soil from Brazil (BRA) and Switzerland (SUI), the Czech Republic (CZE) soil which was previously subjected to intensive in situ bioremediation displayed only negligible changes in community structure. BRA and SUI soil samples showed a clear succession of phylotypes. A rapid response to benzene stress was observed, whereas the response to BTEX pollution was significantly slower. After extended incubation, actinobacterial phylotypes increased in relative abundance, indicating their superior fitness to pollution stress. Commonalities but also differences in the phylotypes were observed. Catabolic gene surveys confirmed the enrichment of actinobacteria by identifying the increase of actinobacterial genes involved in the degradation of pollutants. Proteobacterial phylotypes increased in relative abundance in SUI microcosms after short-term stress with benzene, and catabolic gene surveys indicated enriched metabolic routes. Interestingly, CZE soil, despite staying constant in community structure, showed a change in the catabolic gene structure. This indicates that a highly adapted community, which had to adjust its gene pool to meet novel challenges, has been enriched.

Whole-genome sequencing-based characterization of Streptomyces sp. 6(4): focus on natural product.

We have sequenced the whole genome of Streptomyces sp. 6(4) isolated from tomato roots that presents antifungal activity against phytopathogenic fungi, mainly Bipolaris sorokiniana. The genome has almost 7 Mb and 3368 hypothetical proteins that were analysed and characterized in Uniprot with the emphasis on biological compounds. Multilocus sequence typing (MLST) analyses were performed in an effort to characterize and identify this isolate, resulting in a new sequence type (ST), classified as ST64. Phenetic and phylogenetic trees were constructed to investigate Streptomyces sp. 6(4) evolution and sequence similarity, and the isolate is a strain closer to Streptomyces prasinus and Streptomyces viridosporus . It is known that the genus Streptomyces possess huge metabolic capacity with the presence of cryptic genes. These genes are usually present in clusters, which are responsible for the production of diverse natural products, mainly antibiotics. In addition, 6(4) showed 11 biosynthetic gene clusters through antiSMASH, including 3 polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) type clusters.

Escherichia coli carrying blaNDM-1 obtained from a migratory penguin (Spheniscus magellanicus) in the Brazilian seacoast.

The reservoirs for NDM-producing Enterobacterales are increasing, not only in hospitals, but also in the environment and in the community, challenging the therapeutic efficacy of carbapenems. We aimed to characterize an isolate of Escherichia coli harboring the blaNDM-1 gene recovered from the bloodstream of a penguin (Spheniscus magellanicus) in Southern Brazil. A total of 74 bacterial isolates recovered from arterial blood samples from dead birds were submitted to species identification and antibiotic susceptibility evaluation. One isolate presented resistance to carbapenems (E. coli 89PenNDM) and proved to harbor the blaNDM-1 gene by multiplex high-resolution melting real-time PCR (PCR-HRM). Conjugation experiments indicated that the blaNDM-1 was transmissible to E. coli J53. Whole genome sequencing (WGS) confirmed the presence of the blaNDM-1 gene in a conjugative plasmid (IncA/C2 plasmid) in both the E. coli 89PenNDM and its transconjugants. The isolate was classified as ST 156 and many other resistance genes (e.g., sul1, sul,2, strA, floR, tet(A)) were identified, all carried in the same IncA/C2 plasmid. This is the first report of blaNDM-1-producing E. coli isolated from a penguin in the Brazilian seacoast. The presence of a carbapenemase gene in wildlife animals is of concern as they may become reservoirs of multidrug-resistant bacteria and disseminate them to the environment.

Antibacterial and synergistic activity of a new 8-hydroxyquinoline derivative against methicillin-resistant Staphylococcus aureus.

Aim: To evaluate the antibacterial and synergistic effect of a new 8-hydroxyquinoline derivative (PH176) against MRSA. Materials & methods: PH176 activity was determined by broth microdilution against 38 Staphylococcus aureus clinical isolates. The antibacterial and synergistic effects with oxacillin and nitroxoline were evaluated by time-kill assays to five MRSA isolates. Toxicity was evaluated by in vitro and ex vivo models. Results: The MIC50 and MIC90 of PH176 were 16 and 32 µg/ml, respectively. The PH176 and nitroxoline led to a reduction in colony count for four isolates and the combination of PH176 and oxacillin acted synergically for three isolates. Furthermore, PH176 was determined to be noncytotoxic/nonirritant. Conclusion: These results demonstrate that PH176 has revealed promising results to be a potential candidate to treat MRSA infections.

SARS-CoV-2 nucleic acid shedding is variable for every person.

INTRODUCTION: Considering the persistent positivity on RT-qPCR tests, the results of SARS-CoV-2 were monitored to evaluate the viral RNA shedding period. METHODS: Between March and June 2020, the sequential results of 29 healthcare workers' were monitored using RT-qPCR. RESULTS: More than 50% of the individuals remained RT-qPCR positive after 14 days. Furthermore, this is the first study to describe positive RT-qPCR for SARS-CoV-2 in a healthcare worker with mild symptoms 95 days after the first positive test. CONCLUSIONS: Sequential RT-qPCR results were heterogeneous, and the viral RNA shedding period is unique for each person.

NDM-1-encoding plasmid in Acinetobacter chengduensis isolated from coastal water.

BACKGROUND: Acinetobacter spp. may cause difficult-to-treat nosocomial infections due to acquisition of carbapenemases, including New Delhi metallo-ß-lactamase (NDM). This genus has been pointed out as a possible actor in the early dissemination of blaNDM, and this gene has been documented in a variety of species. OBJECTIVE: Here we describe an Acinetobacter chengduensis (isolate FL51) carrying blaNDM recovered from coastal water in Brazil. METHODS: In vitro techniques included antimicrobial susceptibility and minimum inhibitory concentration tests, PCR, plasmid profile and matting-out/transformation assays. In silico approaches comprised comparative genomic analyses using appropriate databases. RESULTS: FL51 grew at room temperature in a variety of culture media, excluding MacConkey. It showed resistance to all beta-lactams tested and to ciprofloxacin. blaNDM-1 was identified, and a single replicon was observed in plasmid profile. In silico DNA hybridization revealed Acinetobacter FL51 as being Acinetobacter chengduensis. blaNDM-1 was flanked upstream by ISAba14-aphA6-ISAba125 and downstream by bleMBL-trpF-Δtat, inserted in a 41,068 bp non typeable plasmid named pNDM-FL51. This replicon showed high coverage and identity with other sequences present in plasmids deposited on the GenBank database, recovered almost exclusively from Acinetobacter spp., associated with hospital settings and animal sources. CONCLUSION: We described a recently described environmental Acinetobacter species carrying a plasmid-borne blaNDM associated with a Tn125-like structure. Our findings suggest that replicon may play an important role in blaNDM dissemination among distinct settings within this genus and may support the theory of blaNDM emergence from an environmental Acinetobacter.

RT-qPCR half-reaction optimization for the detection of SARS-CoV-2.

INTRODUCTION: The main laboratory test for the diagnosis of coronavirus disease 2019 (COVID-19) is the reverse transcription real-time polymerase chain reaction (RT-qPCR). However, RT-qPCR is expensive because of the number of tests required. This study aimed to evaluate an alternative to the RT-qPCR approach for the detection of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is half of the total volume currently recommended by the US Centers for Disease Control and Prevention. METHODS: The analytical limit of detection (LoD) and the reaction efficiency using half volumes of the RT-qPCR assay were evaluated for the N1 and N2 regions using a synthetic control RNA. A panel of 76 SARS-CoV-2-positive and 26 SARS-CoV-2-negative clinical samples was evaluated to establish clinical sensitivity and specificity. RESULTS: The RT-qPCR assay efficiency was 105% for the half and standard reactions considering the N2 target and 84% (standard) and 101% (half) for N1. The RT-qPCR half-reaction LoD for N1 and N2 were 20 and 80 copies/µL, respectively. The clinical sensitivity and specificity were 100%. The half reaction presented a decrease of up to 5.5 cycle thresholds compared with standard RT-qPCR. CONCLUSIONS: The use of the RT-qPCR half-reaction proved feasible and economic for the detection of SARS-CoV-2 RNA.

Characterization of an emergent high-risk KPC-producing Klebsiella pneumoniae lineage causing a fatal wound infection after spine surgery.

Surgical site infections in instrumented posterior lumbar interbody fusion surgery are normally due to gram-positive bacteria, but gram-negative bacteria can cause infections in cases involving lower lumbar interventions as its closer to the perianal area. Here we report an uncommon fatal wound infection caused by a multidrug-resistant Klebsiella pneumoniae after an elective spine surgery. In silico analysis revealed that LWI_ST16 belonged to ST16, an emergent international clone notable for its increased virulence potential. We also observed that this strain carried a conjugative IncF plasmid encoding resistance genes to beta-lactams (blaKPC-2 and blaOXA-1), tetracycline (tetA), aminoglycosides and fluoroquinolones (aac(6')-Ib-cr). The carbapenemase encoding gene blaKPC-2 was located on a Tn4401e transposon previously characterized to increase blaKPC expression. LWI_ST16 is a strong biofilm producer on polystyrene and capable of forming tower-like structures on a titanium device like the one inserted in the patient's spine. Our findings strengthen the valuable contribution of continuous surveillance of multidrug-resistant and high-risk K. pneumoniae clones to avoid unfavourable clinical outcomes.

Novel mutations in the resistome of a new sequence type (ST262) of clarithromycin resistant Mycobacterium abscessus subsp. massiliense.

OBJECTIVES: The aim of this study was to evaluate the relationship between antimicrobial susceptibility and genotype of a Mycobacterium abscessus subsp. massiliense isolate obtained from the respiratory tract of a patient in southern Brazil. METHODS: The isolate (named Myco1POA) was submitted to whole-genome sequencing using an Illumina MiSeq platform. Data were analysed using Trim Galore!, SPAdes Genome Assembler, Geneious and BioEdit software. Multilocus sequence typing (MLST) was performed by in silico analysis of seven housekeeping genes according to the Institut Pasteur database. The antimicrobial susceptibility profile was determined by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: Several mutations in genes related to antimicrobial resistance were identified in Myco1POA. MLST indicated that the isolate belonged to a novel sequence type (ST), which was designated ST262. Phenotypic susceptibility and genotypic findings were concordant, except for clarithromycin [erm(41) and rrl genes]. CONCLUSION: Here we describe the genome sequence of M. abscessus subsp. massiliense Myco1POA identified as a novel sequence type (ST262) and indicate possible new gene mutations leading to clarithromycin resistance.

Evaluation of the susceptibility test of polymyxin B using the commercial test Policimbac®.

Broth microdilution (BMD), the reference method to determine bacterial susceptibility to polymyxins, is a laborious and time-consuming technique. Policimbac® is a commercial test panel which uses lyophilized polymyxin B to determine the minimum inhibitory concentration for Gram-negative isolates. This study evaluated the performance of Policimbac® in comparison with BMD for 110 isolates. Although the Policimbac® presented a very low essential agreement, the categorical agreement with BMD was optimal. Policimbac® is an alternative approach to BMD for evaluating the susceptibility to polymyxin B.