Understanding how pre- and probiotics affect the gut microbiome and metabolic health.

The gut microbiome, a complex assembly of microorganisms, significantly impacts human health by influencing nutrient absorption, the immune system, and disease response. These microorganisms form a dynamic ecosystem that is critical to maintaining overall well-being. Prebiotics and probiotics are pivotal in regulating gut microbiota composition. Prebiotics nourish beneficial bacteria and promote their growth, whereas probiotics help maintain balance within the microbiome. This intricate balance extends to several aspects of health, including maintaining the integrity of the gut barrier, regulating immune responses, and producing metabolites crucial for metabolic health. Dysbiosis, or an imbalance in the gut microbiota, has been linked to metabolic disorders such as type 2 diabetes, obesity, and cardiovascular disease. Impaired gut barrier function, endotoxemia, and low-grade inflammation are associated with toll-like receptors influencing proinflammatory pathways. Short-chain fatty acids derived from microbial fermentation modulate anti-inflammatory and immune system pathways. Prebiotics positively influence gut microbiota, whereas probiotics, especially Lactobacillus and Bifidobacterium strains, may improve metabolic outcomes, such as glycemic control in diabetes. It is important to consider strain-specific effects and study variability when interpreting these findings, highlighting the need for further research to optimize their therapeutic potential. The aim of this report is therefore to review the role of the gut microbiota in metabolic health and disease and the effects of prebiotics and probiotics on the gut microbiome and their therapeutic role, integrating a broad understanding of physiological mechanisms with a clinical perspective.

The effect of probiotics, prebiotics or synbiotics on metabolic outcomes in individuals with diabetes: a systematic review and meta-analysis.

AIMS/HYPOTHESIS: The aim was to conduct a systematic review and meta-analysis of randomised controlled clinical trials assessing the effect of probiotic, prebiotic or synbiotic supplementation on gut microbiota and glucose control and lipid levels in individuals with diabetes. METHODS: MEDLINE, EMBASE and the Cochrane Library were searched. The eligibility criteria for the studies was involvement of participants with a diagnosis of type 1 or type 2 diabetes. Metabolic outcomes (glucose control, insulinaemia, and lipid profile) of any probiotic, prebiotic or synbiotic supplementation related to modification of gut microbiota (prebiotics, probiotics and synbiotics) were analysed. We provided a narrative synthesis and meta-analysis of the findings on metabolic outcomes from the studies. Metabolic outcomes were extracted post-intervention and expressed as mean differences (MDs) and 95% CIs between treatment and comparator groups. We pooled the results using a random-effects meta-analysis. The meta-analysis was conducted using Review Manager (RevMan) software. RESULTS: After the removal of duplicates and ineligible studies, 5219 studies were retained for review of titles and abstracts. The number of articles was reduced to 130 by review, for which the full-text articles were obtained and reassessed, 38 of which were included in the final meta-analysis. Overall, the use of prebiotics, probiotics or synbiotics reduced HbA1c levels, but did not reach the threshold for significance (-2.17 mmol/mol, 95% CI -4.37, 0.03; p = 0.05, [-0.20%, 95% CI -0.40 to 0.00; p = 0.05, I2 = 66%]) and had no effect on LDL-cholesterol levels (-0.05 mmol/l; 95% CI -0.14, 0.05, p = 0.35, I2 = 37%). However, their consumption decreased levels of fasting blood glucose (-0.58 mmol/l; 95% CI -0.86, -0.30; p < 0.01, I2 = 60%), total cholesterol (-0.14 mmol/l; 95% CI -0.26, -0.02, p = 0.02, I2 = 39%), triacylglycerols (-0.11 mmol/l; 95% CI -0.20, -0.02, p = 0.01, I2= 21%) and insulinaemia (-10.51 pmol/l; 95% CI -16.68,-4.33, p < 0.01, I2 = 74%), and increased HDL-cholesterol levels (0.04 mmol/l; 95% CI 0.01, 0.07, p < 0.01, I2= 24%). CONCLUSIONS/INTERPRETATION: In individuals with diabetes mellitus, supplementation with probiotics, prebiotics or synbiotics improved metabolic variables, although the magnitude of this effect is low. Our results suggest that consumption of probiotics, prebiotics or synbiotics may be a potential adjuvant treatment for improving metabolic outcomes. REGISTRATION: PROSPERO ID CRD42017080071. Graphical abstract.

Molecular investigation of isolates from a multistate polymicrobial outbreak associated with contaminated total parenteral nutrition in Brazil.

BACKGROUND: Between November 2013 and June 2014, 56 cases of bacteremia (15 deaths) associated with the use of Total Parenteral Nutrition (TPN) and/or calcium gluconate (CG) were reported in four Brazilian states. METHODS: We analyzed 73 bacterial isolates from four states: 45 from blood, 25 from TPN and three from CG, originally identified as Acinetobacter baumannii, Rhizobium radiobacter, Pantoea sp. or Enterobacteriaceae using molecular methods. RESULTS: The first two bacterial species were confirmed while the third group of species could not be identified using standard identification protocols. These isolates were subsequently identified by Multi-Locus Sequence Analysis as Phytobacter diazotrophicus, a species related to strains from similar outbreaks in the United States in the 1970's. Within each species, TPN and blood isolates proved to be clonal, whereas the R. radiobacter isolates retrieved from CG were found to be unrelated. CONCLUSION: This is the first report of a three-species outbreak caused by TPN contaminated with A. baumannii, R. radiobacter and P. diazotrophicus. The concomitant presence of clonal A. baumannii and P. diazotrophicus isolates in several TPN and blood samples, as well as the case of one patient, where all three different species were isolated simultaneously, suggest that the outbreak may be ascribed to a discrete contamination of TPN. In addition, this study highlights the clinical relevance of P. diazotrophicus, which has been involved in outbreaks in the past, but was often misidentified as P. agglomerans.

PCR Assay Based on the gyrB Gene for Rapid Identification of Acinetobacter baumannii-calcoaceticus Complex at Specie Level.

BACKGROUND: The genus Acinetobacter sp. comprises more than 50 species, and four are closely related and difficult to be distinguished by either phenotypic or genotypic methods: the Acinetobacter calcoaceticus-baumannii complex (ABC). The correct identification at species level is necessary mainly due to the epidemiological aspects. METHODS: We evaluated a multiplex PCR for gyrB gene to identify the species of the ABC using the sequencing of the ITS 16S-23S fragment as a gold standard. Isolates identified as Acinetobacter calcoaceticus-baumannii from three hospitals at southern Brazil in 2011 were included in this study. RESULTS: A total of 117 isolates were obtained and 106 (90.6%) were confirmed as A. baumannii, 6 (5.1%) as A. nosocomialis and 4 (3.4%) as A. pittii by PCR for gyrB gene. Only one isolate did not present a product of the PCR for the gyrB gene; this isolate was identified as Acinetobacter genospecie 10 by sequencing of ITS. We also noted that the non-A. baumannii isolates were recovered from respiratory tract (8/72.7%), blood (2/18.2%) and urine (1/9.1%), suggesting that these species can cause serious infection. CONCLUSION: These findings evidenced that the multiplex PCR of the gyrB is a feasible and simple method to identify isolates of the ABC at the species level.

Emergence of Acinetobacter baumannii ST730 carrying the blaOXA-72 gene in Brazil.

Over the last decade, Acinetobacter baumannii resistant to carbapenems has emerged in many medical centres and has been commonly associated with high morbimortality. In Brazil, this resistance is mainly attributed to the spread of OXA-23-producing clones and, to a lesser extent, to OXA-143-producing clones. Here, we describe, for the first time, two OXA-72-producing A. baumannii isolates in southern Brazil to a broad spectrum of antibiotics, except polymyxin B and tigecycline. Molecular typing by multilocus sequence typing (MLST) demonstrated that both OXA-72-producing isolates belong to a new sequence type (ST), ST730, which was recently identified in OXA-23-producing A. baumannii isolates in São Paulo, Brazil. We demonstrate that the two A. baumannii ST730 isolates carrying blaOXA-72share a common ancestral origin with the blaOXA-23producers in Brazil. This observation reinforces the importance of strain-typing methods in order to clarify the dynamics of the emergence of new clones in a geographic region.

Molecular typing of mcr-1 Escherichia coli isolates from pigs and farm environment based on fumC and fimH alleles.

Background: The dissemination of polymyxin resistance represents a significant threat to public health. Materials & methods: Sequence-based typing was performed by 53 mcr-1 Escherichia coli isolates using fumC/fimH (CH) genes to characterize clones spreading from pig farming. Furthermore, 12 isolates had their whole genome sequenced for phylogenetic study. Results: The isolates were classified into 22 distinct CH types, and two novel CH types (CH41-1578 and CH4-1579) and one sequence type (ST12652) was also described. According to phylogenetic study, both multilocus sequence typing and CH methods grouped the isolates similarly. Conclusion: Our findings suggest that the dissemination of the mcr-1 gene in pig farming has occurred mainly by horizontal gene transfer, and CH typing proved to be a good tool to characterize E. coli clones.

Validation of a MALDI-TOF MS Method for SARS-CoV-2 Detection on the Bruker Biotyper and Nasopharyngeal Swabs: A Brazil-UK Collaborative Study.

We developed a MALDI-TOF mass spectrometry method for the detection of the SARS-CoV-2 virus in saliva-gargle samples using Shimadzu MALDI-TOF mass spectrometers in the UK. This was validated in the USA to CLIA-LDT standards for asymptomatic infection detection remotely via sharing protocols, shipping key reagents, video conferencing, and data exchange. In Brazil, more so than in the UK and USA, there is a need to develop non-PCR-dependent, rapid, and affordable SARS-CoV-2 infection screening tests that also identify variant SARS-CoV-2 and other virus infections. In addition, travel restrictions necessitated remote collaboration with validation on the available clinical MALDI-TOF-the Bruker Biotyper (microflex® LT/SH)-and on nasopharyngeal swab samples, as salivary gargle samples were not available. The Bruker Biotyper was shown to be almost log103 more sensitive at the detection of high molecular weight spike proteins. A protocol for saline swab soaks out was developed, and duplicate swab samples collected in Brazil were analyzed by MALDI-TOF MS. The swab collected sample spectra that varied from that of saliva-gargle in three additional mass peaks in the mass region expected for IgG heavy chains and human serum albumin. A subset of clinical samples with additional high mass, probably spike-related proteins, were also found. Further, spectral data comparisons and analysis, subjected to machine learning algorithms in order to resolve RT-qPCR positive from RT-qPCR negative swab samples, showed 56-62% sensitivity, 87-91% specificity, and a 78% agreement with RT-qPCR scoring for SARS-CoV-2 infection.

The impact of dietary, surgical, and pharmacological interventions on gut microbiota in individuals with diabetes mellitus: A systematic review.

AIMS: To conduct a systematic review assessing the association between dietary, surgical, and pharmacological interventions and changes in the gut microbiota of individuals with diabetes. METHODS: The MEDLINE, EMBASE, and Cochrane Library databases were searched focusing on the effects of dietary, bariatric surgery, and pharmacological interventions on gut microbiota in adults with diabetes. Studies were classified based on qualitative changes using a simple vote-counting method, evaluating reduction, no effect, or an increase in the gut microbiota outcomes. RESULTS: 6,004 studies were retained to review their titles and abstracts. A total of 149 full-text articles were reassessed, of which 49 were included in the final analysis. This review indicates that dietary, surgical, and pharmacological interventions increase or decrease bacterial populations from more than 60 families, genera, or species. In general, the interventions led to an increase in the bacterial population from phylum Firmicutes, mainly Lactobacillus species, compared to the gram-negative bacterial population from phylum Bacteroidetes. CONCLUSIONS: The results of the included studies suggest that interventions aimed at reducing species related to uncontrolled diabetes and increasing species related to the healthy gut are potential adjuvants in treating diabetes; however, well-conducted interventional studies targeting gut microbiota are necessary.

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.

High-performance method to detection of Klebsiella pneumoniae Carbapenemase in Enterobacterales by LC-MS/MS.

Carbapenem-resistant Enterobacterales (CREs) have been recognized as an important threat to global health. CRE cause the majority of the difficult-to-treat infections in health-care settings and are associated with high mortality. Klebsiella pneumoniae carbapenemase (KPC)-producing CREs, in particular Klebsiella pneumoniae, are globally disseminated and responsible for a large number of outbreaks. Development of rapid methods for KPC detection can provide great clinical and epidemiological benefits to prevent KPC dissemination. The aim of this study was to standardize and validate a LC-MS/MS method to detect KPC. This method was also tested against a broad variety of species, including CRE with other carbapenemase genes and the recently reported mcr-1. For validation, 111 isolates with reduced susceptibility to carbapenems were selected (49 KPC-positive and 62 KPC-negative). The presence of four tryptic peptides related to the KPC enzyme was evaluated, and the identification of at least two of them classified the isolate as "KPC-positive." The LTLGSALAAPQR and LALEGLGVNGQ peptides were both detected in 47 of 49 isolates with the blaKPC gene. The other two peptides, GFLAAAVLAR and APIVLAVYTR, were detected in 46 and 19 isolates with the blaKPC gene, respectively. The method correctly classified 47 of 49 KPC-positive and all KPC-negative isolates yielding 96.07% of sensitivity and 100% of specificity. In conclusion, our results demonstrate that the KPC peptide markers were robustly detected by the method which presented high sensitivity and full specificity and therefore can be used as a reliable method to identify this resistance mechanism.

Genetic relatedness, Virulence factors and Antimicrobial Resistance of C. difficile strains from hospitalized patients in a multicentric study in Brazil.

BACKGROUND: Clostridium (Clostridioides) difficile infection (CDI) is recognized worldwide as a public health concern, related mainly with hypervirulent strains. In Brazil there are few studies about molecular epidemiology of C. difficile, for this reason, we aimed to characterize C. difficile isolates from a large cohort study of three different Brazilian states to identify virulence and resistance genes, specifically genes related to metronidazole and vancomycin resistance. METHODS: All 153 fecal samples were submitted to C. difficile culture in CM0601 broth. Identification of suspected colonies was confirmed by matrix-assisted laser desorption/ionization (MALDI-TOF/MS, Brucker Daltonics, Germany). The tcdA and tcdB toxin were searched by PCR. The sequence type (ST) was determinate by multilocus sequencing typing (MLST) and susceptibility profile was performed by agar dilution method. RESULTS: Among the 16 isolates, we identified fourteen different STs, five belonging to Clade 1, one to Clade 2 and eight news STs with high similarity levels. Resistance (ermB, tetM, VanW and nimB) and virulence genes (cwp84, cwp66, cwp2, fbpA and secA) were found in toxigenic strains. CONCLUSION: Differently from other studies, we found high levels of resistance to vancomycin. These results suggest that the main circulating strains in Brazil belong to Clade 1 and have high pathogenicity and resistance profile.

Comparative Analysis of Carbapenem-Resistant Acinetobacter baumannii Sequence Types in Southern Brazil: From the First Outbreak (2007-2008) to the Endemic Period (2013-2014).

Acinetobacter baumannii is considered an important pathogen of clinical significance that is responsible for a wide range of nosocomial infections. Carbapenem resistance among A. baumannii isolates has increased dramatically in the Past years because of the emergence and dissemination of specific epidemic clones. We aimed to characterize the population structure of A. baumannii isolates from Porto Alegre city, Southern Brazil, in two distinct periods: during the first carbapenem-resistant A. baumannii (CRAB) outbreak (2007-2008) and 5 years later when the CRAB reached endemic levels (2013-2014). The study included 49 CRAB isolates collected in two periods: 2007-2008 (31 isolates) and 2013-2014 (18 isolates). Multilocus sequence typing (MLST) was performed according to Institute Pasteur, followed by eBURST analysis. PCR was used to detect integrase gene, blaNDM, and oxacillinase genes, and also to detect the ISAba1 element upstream blaOXA-23. The eBURST analysis identified the clonal complexes (CCs) CC15, CC32, CC79, CC216, CC221, and CC464 in the first period (2007-2008) and CC1, CC2, CC15, CC79, and CC162 during the endemic period (2013-2014). Molecular analysis by MLST identified 13 new sequence types. As we found A. baumannii with the blaOXA-23 gene of several CCs, it can be concluded that the increase of CRAB infections are not related to a specific clone. Furthermore, the high-risk CC15 and CC79 were related to the first CRAB outbreak and these CCs persisted up to 2014 in Porto Alegre city. The international clones CC1 and CC2 were observed for the first time in only the second period (2013-2014), alerting to the emergence of these clones in Southern Brazil.

In vitro antimicrobial activity of imipenem plus amikacin or polymyxin B against carbapenem-resistant Pseudomonas aeruginosa isolates.

The aim of this study was to evaluate the antimicrobial activity of imipenem plus amikacin or polymyxin B against six carbapenem-resistant P. aeruginosa by time-kill assay. Synergism was observed in two isolates for combinations with amikacin. For combinations with polymyxin B, synergism and antagonism also occurred in two isolates.

Clostridium difficile contamination in retail meat products in Brazil.

INTRODUCTION: Clostridium difficile is an important cause of diarrhoea, particularly in patients receiving antibiotic therapy. Recent studies have shown that a substantial proportion of C. difficile infections are acquired in the community, as a zoonotic disease. Brazil is a large exporter of meat and so far no study has evaluated meat contamination with C. difficile spores. METHODS: Here we analysed 80 retail meat products purchased from local supermarkets in a Brazilian metropolis (Porto Alegre, Southern Brazil). Samples from these products were grown in anaerobic conditions, and tested with a real time polymerase chain reaction test. RESULTS: Contamination with C. difficile spores was not found in the study. Bacteria isolated from meat included Streptococcus gallolyticus, Lactobacillus plantarum, Enterococcus gallinarum and Pediococcus acidilactici. DISCUSSION: Close vigilance is required in order to guarantee the quality of Brazilian retail meat in the long term.

First detection of Pseudomonas aeruginosa ST2963 from hospital effluent: A draft genome analysis.

OBJECTIVES: Pseudomonas aeruginosa strains L25 and M12 were isolated from hospital effluent in southern Brazil. METHODS: The whole genomes of the isolates were sequenced using an Illumina MiSeq system. The data were analysed using SPAdes, Prokka and Geneious, and antimicrobial resistance genes were predicted using ResFinder. PubMLST protocols were used to define the sequence type (ST). RESULTS: Many multidrug efflux pump systems as well as various antimicrobial resistance genes were identified in the two P. aeruginosa strains. The strains were identified as ST2963, a novel carbapenem-resistant sequence type. CONCLUSIONS: Here we describe the genome sequences of two carbapenem-resistant P. aeruginosa strains and characterised a novel sequence type (ST2963).

Clostridium difficile contamination in retail meat products in Brazil

ABSTRACT Introduction Clostridium difficile is an important cause of diarrhoea, particularly in patients receiving antibiotic therapy. Recent studies have shown that a substantial proportion of C. difficile infections are acquired in the community, as a zoonotic disease. Brazil is a large exporter of meat and so far no study has evaluated meat contamination with C. difficile spores. Methods Here we analysed 80 retail meat products purchased from local supermarkets in a Brazilian metropolis (Porto Alegre, Southern Brazil). Samples from these products were grown in anaerobic conditions, and tested with a real time polymerase chain reaction test. Results Contamination with C. difficile spores was not found in the study. Bacteria isolated from meat included Streptococcus gallolyticus, Lactobacillus plantarum, Enterococcus gallinarum and Pediococcus acidilactici. Discussion Close vigilance is required in order to guarantee the quality of Brazilian retail meat in the long term.

Evaluation of heteroresistance to polymyxin B among carbapenem-susceptible and -resistant Pseudomonas aeruginosa.

One hundred and twenty-four Pseudomonas aeruginosa isolates were selected for antimicrobial susceptibility testing with anti-pseudomonal agents, MIC determination for polymyxin B and metallo-beta-lactamase detection (genes blaSPM, blaVIM-1, blaNDM-1 and blaIMP). According to the imipenem and/or meropenem susceptibility profile, a set of randomly selected isolates (12 isolates carbapenem-susceptible and 12 isolates carbapenem-resistant) were evaluated for heteroresistance to polymyxin B. Heteroresistance testing was performed by plating the isolates onto increasing concentrations of polymyxin B (from 0 to 8.0 mg l(-1)). The population analysis profile (PAP) was defined as the ratio of the number of colony-forming units on the plate with the highest concentration of polymyxin B at which bacterial growth occurred against the number of colony-forming units on the plate without antibiotic. Isolates presenting subpopulations that exhibited growth at polymyxin B concentrations ≥2 mg l(-1) were considered heteroresistant. Isolates containing subpopulations that grew at polymyxin B concentrations at least twice as high as the original MIC but <2 mg l(-1) were considered heterogeneous. Antimicrobial susceptibility testing results indicated a variable degree of susceptibility: high levels of resistance to gentamicin (30.6 %) and imipenem (29.0 %); low levels of resistance to aztreonam (1.6 %) and ciprofloxacin (4.8 %). All isolates were susceptible to polymyxin B: MIC50 and MIC90 were 1 mg l(-1) and 2 mg l(-1), respectively. Thirty-seven isolates (30 %) were carbapenem-resistant. Four isolates resistant to carbapenems were positive for blaIMP. There were no heteroresistant subpopulations in the carbapenem-susceptible group, but three isolates presented heterogeneous subpopulations. The PAP frequency ranged from 2.1×10(-4) to 6.9×10(-8). In the carbapenem-resistant group, one isolate was heteroresistant. Six isolates in this group presented heterogeneous subpopulations. In the resistant population, the PAP frequency ranged from 2.1×10(-7) to 2.6×10(-4). In this study, polymyxin B heteroresistance in P. aeruginosa was uncommon and occurred in only one carbapenem-resistant isolate, despite the fact that several isolates presented heterogeneous subpopulations with increased polymyxin B MICs.