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Abstract Background Breastmilk is a dynamic fluid whose initial function is to provide the most adapted nutrition to the neonate. Additional attributes have been recently ascribed to breastmilk, with the evidence of a specific microbiota and the presence of various components of the immune system, such as cytokines and leukocytes. The composition of breastmilk varies through time, according to the health status of mother and child, and altogether contributes to the future health of the infant. Obesity is a rising condition worldwide that creates a state of systemic, chronic inflammation including leukocytosis. Here, we asked whether colostrum, the milk produced within the first 48 h post-partum, would contain a distinct leukocyte composition depending on the body mass index (BMI) of the mother. Methods We collected peripheral blood and colostrum paired samples from obese (BMI > 30) and lean (BMI < 25) mothers within 48 h post-partum and applied a panel of 6 antibodies plus a viability marker to characterize 10 major leukocyte subpopulations using flow cytometry. Results The size, internal complexity, and surface expression of CD45 and CD16 of multiple leukocyte subpopulations were selectively regulated between blood and colostrum irrespective of the study groups, suggesting a generalized cell-specific phenotype alteration. In obesity, the colostrum B lymphocyte compartment was significantly reduced, and CD16+ blood monocytes had an increased CD16 expression compared to the lean group. Conclusions This is the first characterization of major leukocyte subsets in colostrum of mothers suffering from obesity and the first report of colostrum leukocyte subpopulations in Latin America. We evidence various significant alterations of most leukocyte populations between blood and colostrum and demonstrate a decreased colostrum B lymphocyte fraction in obesity. This pioneering study is a stepping stone to further investigate active immunity in human breastmilk.

IntroductionTuberculosis (TB) is among the deadliest diseases worldwide, and its impact is mainly due to the continuous emergence of resistant isolates during treatment due to the laborious process of resistance diagnosis, nonadherence to treatment and circulation of previously resistant isolates of Mycobacterium tuberculosis. In this study, we evaluated the performance and functionalities of web-based tools, including Mykrobe, TB-profiler, PhyResSE, KvarQ, and SAM-TB, for detecting resistance in 88 Ecuadorian isolates of Mycobacterium tuberculosis drug susceptibility tested previously. Statistical analysis was used to determine the correlation between genomic and phenotypic analysis. Our results showed that with the exception of KvarQ, all tools had the highest correlation with the conventional drug susceptibility test (DST) for global resistance detection (98% agreement and 0.941 Cohen's kappa), while SAM-TB, PhyResSE, TB-profiler and Mykrobe had better correlations with DST for first-line drug analysis individually. We also identified that in our study, only 50% of mutations characterized by the web-based tools in the rpoB, katG, embB, pncA, gyrA and rrs regions were canonical and included in the World Health Organization (WHO) catalogue. Our findings suggest that SAM-TB, PhyResSE, TB-profiler and Mykrobe were efficient in determining canonical resistance-related mutations, but more analysis is needed to improve second-line detection. Improving surveillance programs using whole-genome sequencing tools for first-line drugs, MDR-TB and XDR-TB is essential to understand the molecular epidemiology of TB in Ecuador.ImportanceTuberculosis, an infectious disease caused by Mycobacterium tuberculosis, most commonly affects the lungs and is often spread through the air when infected people cough, sneeze, or spit. However, despite the existence of effective drug treatment, patient adherence, long duration of treatment, and late diagnosis have reduced the effectiveness of therapy and increased drug resistance. The increase in resistant cases, added to the impact of the COVID-19 pandemic, has highlighted the importance of implementing efficient and timely diagnostic methodologies worldwide. The significance of our research is in evaluating and identifying a more efficient and user-friendly web-based tool to characterize resistance in Mycobacterium tuberculosis by whole-genome sequencing, which will allow more routine application to improve TB strain surveillance programs locally.

Genomics has significantly revolutionized pathogen surveillance, particularly in epidemiological studies, the detection of drug-resistant strains, and disease control. Despite its potential, the representation of Latin American countries in the genomic catalogues of Mycobacterium tuberculosis (Mtb), the bacteria responsible for Tuberculosis (TB), remains limited. In this study, we present a whole genome sequencing (WGS)-based analysis of 85 Mtb clinical strains from 17 Mexican states, providing insights into local adaptations and drug resistance signatures in the region. Our results reveal that the Euro-American lineage (L4) accounts for 94% of our dataset, showing 4.1.2.1 (Haarlem, n = 32), and 4.1.1.3 (X-type, n = 34) sublineages as the most prevalent. We report the presence of the 4.1.1.3 sublineage, which is endemic to Mexico, in six additional locations beyond previous reports. Phenotypic drug resistance tests showed that 34 out of 85 Mtb samples were resistant, exhibiting a variety of resistance profiles to the first-line antibiotics tested. We observed high levels of discrepancy between phenotype and genotype associated with drug resistance in our dataset, including pyrazinamide-monoresistant Mtb strains lacking canonical variants of drug resistance. Expanding the Latin American Mtb genome databases will enhance our understanding of TB epidemiology and potentially provide new avenues for controlling the disease in the region.

Abstract Whole genome sequencing (WGS) has been shown to be superior to traditional procedures of genotyping in tuberculosis (TB), nevertheless, reports of its use in drug resistant TB (DR-TB) isolates circulating in Mexico, are practically unknown. Considering the above the main of this work was to identify and characterize the lineages and genomic transmission clusters present in 67 DR-TB isolates circulating in southeastern Mexico. The results show the presence of three major lineages: L1 (3%), L2 (3%) and L4 (94%), the last one included 16 sublineages. Sublineage 4.1.1.3 (X3) was predominant in 18 (27%) of the isolates, including one genomic cluster, formed by eleven multidrug resistant isolates and sharing the SIT 3278, which seems to be restricted to Mexico. By the use of WGS, it was possible to identify the high prevalence of L4 and a high number of sublineages circulating in the region, also was recognized the presence of a novel X3 sublineage, formed exclusively by multidrug resistant isolates and with restrictive circulation in Mexico for at least the past 17 years.

Abstract Ulcerative colitis (UC) is a frequent type of inflammatory bowel disease, characterized by periods of remission and exacerbation. Gut dysbiosis may influence pathophysiology and clinical response in UC. The purpose of this study was to evaluate whether gut microbiota is related to the active and remission phases of pancolitis in patients with UC as well as in healthy participants. Fecal samples were obtained from 18 patients with UC and clinical‐endoscopic evidenced pancolitis (active phase n = 9 and remission phase n = 9), as well as 15 healthy participants. After fecal DNA extraction, the 16S rRNA gene was amplified and sequenced (Illumina MiSeq), operational taxonomic units were analyzed with the QIIME software. Gut microbiota composition revealed a higher abundance of the phyla Proteobacteria and Fusobacteria in active pancolitis, as compared with remission and healthy participants. Likewise, a marked abundance of the genus Bilophila and Fusobacteria were present in active pancolitis, whereas a higher abundance of Faecalibacterium characterized both remission and healthy participants. LEfSe analysis showed that the genus Roseburia and Faecalibacterium were enriched in remission pancolitis, and genera Bilophila and Fusobacterium were enriched in active pancolitis. The relative abundance of Fecalibacterium and Roseburia showed a higher correlation with fecal calprotectin, while Bilophila and Fusobacterium showed AUCs (area under the curve) of 0.917 and 0.988 for active vs. remission pancolitis. The results of our study highlight the relation of gut dysbiosis with clinically relevant phases of pancolitis in patients with UC. Particularly, Fecalibacterium, Roseburia, Bilophila, and Fusobacterium were identified as genera highly related to the different clinical phases of pancolitis.

Chronic wounds are a major health problem that cause millions of dollars in expenses every year. Among all the treatments used, active wound treatments such as enzymatic treatments represent a cheaper and specific option with a fast growth category in the market. In particular, bacterial and plant proteases have been employed due to their homology to human proteases, which drive the normal wound healing process. However, the use of these proteases has demonstrated results with low reproducibility. Therefore, alternative sources of proteases such as snake venom have been proposed. Here, we performed a functional mining of proteases from rattlesnakes (Crotalus ornatus, C. molossus nigrescens, C. scutulatus, and C. atrox) due to their high protease predominance and similarity to native proteases. To characterize Crotalus spp. Proteases, we performed different protease assays to measure and confirm the presence of metalloproteases and serine proteases, such as the universal protease assay and zymography, using several substrates such as gelatin, casein, hemoglobin, L-TAME, fibrinogen, and fibrin. We found that all our venom extracts degraded casein, gelatin, L-TAME, fibrinogen, and fibrin, but not hemoglobin. Crotalus ornatus and C. m. nigrescens extracts were the most proteolytic venoms among the samples. Particularly, C. ornatus predominantly possessed low molecular weight proteases (P-I metalloproteases). Our results demonstrated the presence of metalloproteases capable of degrading gelatin (a collagen derivative) and fibrin clots, whereas serine proteases were capable of degrading fibrinogen-generating fibrin clots, mimicking thrombin activity. Moreover, we demonstrated that Crotalus spp. are a valuable source of proteases that can aid chronic wound-healing treatments.

Oligosaccharides are significant in mammalian milk, where they serve as prebiotics that promote the growth of beneficial gut bacteria in infants. Comprehensive research of milk oligosaccharides requires precise and validated analytical methods for compositional studies. To address this need, the focus of our study was to develop and validate an analytical method using UPLC-MS/MS to quantify seven specific oligosaccharides found in mammalian milk. The developed and optimized method has adequate linearity, accuracy, and precision parameters. The detection (LOD) and quantification (LOQ) limits for the seven compounds ranged from 0.0018 to 0.0030 μg/mL and 0.0054–0.0063 μg/mL, respectively. The sample preparation method yielded recovery rates above 90.5 %. Furthermore, no significant matrix effect was observed. The validated method was successfully applied to human, goat, and bovine milk samples, demonstrating its proficiency in identifying variances in the concentration of oligosaccharides across different mammals. This versatile method will allow future research about factors affecting oligosaccharide composition.

The natural products (NPs) biosynthetic gene clusters (BGCs) represent the adapting biochemical toolkit for microorganisms to thrive different microenvironments. Despite their high diversity, particularly at the genomic level, detecting them in a shake-flask is challenging and remains the primary obstacle limiting our access to valuable chemicals. Studying the molecular mechanisms that regulate BGC expression is crucial to design of artificial conditions that derive on their expression. Here, we propose a phylogenetic analysis of regulatory elements linked to biosynthesis gene clusters, to classify BGCs to regulatory mechanisms based on protein domain information. We utilized Hidden Markov Models from the Pfam database to retrieve regulatory elements, such as histidine kinases and transcription factors, from BGCs in the MIBiG database, focusing on actinobacterial strains from three distinct environments: oligotrophic basins, rainforests, and marine environments. Despite the environmental variations, our isolated microorganisms share similar regulatory mechanisms, suggesting the potential to activate new BGCs using activators known to affect previously characterized BGCs.

Genetic variation in tuberculosis is influenced by the host environment, patients with comorbidity, and tuberculosis–type 2 diabetes mellitus (TB-T2DM) and implies a higher risk of treatment failure and development of drug resistance. Considering the above, this study aimed to evaluate the influence of T2DM on the dynamic of polymorphisms related to antibiotic resistance in TB. Fifty individuals with TB-T2DM and TB were initially characterized, and serial isolates of 29 of these individuals were recovered on day 0 (diagnosis), 30, and 60. Genomes were sequenced, variants related to phylogeny and drug resistance analyzed, and mutation rates calculated and compared between groups. Lineage X was predominant. At day 0 (collection), almost all isolates from the TB group were sensitive, apart from four isolates from the TB-T2DM group showing the mutation katG S315T, from which one isolate had the mutations rpoB S450L, gyrA A90G, and gyrA D94G. This pattern was observed in a second isolate at day 30. The results provide a first overview of the dynamics of mutations in resistance genes from individuals with TB-T2DM, describing an early development of resistance to isoniazid and a rapid evolution of resistance to other drugs. Although preliminary, these results help to explain the increased risk of drug resistance in individuals with TB and T2DM.

Abstract Snake venoms are rich sources of proteins with potential biotechnological and pharmaceutical applications. Among them, metalloproteases (MPs) and phospholipases A2 (PLA2) are the most abundant. Their isolation involves a multistep chromatographic approach, which has proven to be effective, however implies high operating costs and long processing times. In this study, a cost-effective and simple method based on aqueous two-phase systems (ATPS) was developed to recover MPs and PLA2 from Crotalus molossus nigrescens venom. A system with PEG 400 g mol−1, volume ratio (VR) 1, tie line length (TLL) 25% w/w and pH 7 showed the best performance for PLA2 recovery. In systems with PEG 400 g mol−1, VR 1, TLL 15% w/w, pH 7 and 1 and 3% w/w of NaCl, selective recovery of MP subtype P-III was achieved; whereas, in a system with PEG 400 g mol−1, VR 1, TLL 25% w/w and pH 8.5, MP subtypes P-I and P-III were recovered. Due to their low costs, ethanol–salt systems were also evaluated, however, failed to differentially partition PLA2 and MPs. The use of ATPS could contribute to the simplification and cost reduction of protein isolation processes from snake venoms and other toxin fluids, as well as potentially aid their biochemical, proteomic and biological analyses. Graphic Abstract

Abstract Given its biocompatibility, rheological, and physiological properties, hyaluronic acid (HA) has become a biomaterial of increasing interest with multiple applications in medicine and cosmetics. In recent decades, microbial fermentations have become an important source for the industrial production of HA. However, due to its final applications, microbial HA must undergo critical and long purification processes to ensure clinical and cosmetic grade purity. Aqueous two-phase systems (ATPS) have proven to be an efficient technique for the primary recovery of high-value biomolecules. Nevertheless, their implementation in HA downstream processing has been practically unexplored. In this work, polyethylene glycol (PEG)–citrate ATPS were used for the first time for the primary recovery of HA produced with an engineered strain of Streptococcus equi subsp. zooepidemicus. The effects of PEG molecular weight (MW), tie-line length (TLL), volume ratio (VR), and sample load on HA recovery and purity were studied with a clarified fermentation broth as feed material. HA was recovered in the salt-rich bottom phase, and its recovery increased when a PEG MW of 8000 g mol−1 was used. Lower VR values (0.38) favoured HA recovery, whereas purity was enhanced by a high VR (3.50). Meanwhile, sample load had a negative impact on both recovery and purity. The ATPS with the best performance was PEG 8000 g mol−1, TLL 43% (w/w), and VR 3.50, showing 79.4% HA recovery and 74.5% purity. This study demonstrated for the first time the potential of PEG–citrate ATPS as an effective primary recovery strategy for the downstream process of microbial HA.

Hyaluronic acid (HA) is a biopolymer with a wide range of applications, mainly in the cosmetic and pharmaceutical sectors. Typical industrial-scale production utilizes organisms that generate HA during their developmental cycle, such as Streptococcus equi sub. zooepidemicus. However, a significant disadvantage of using Streptococcus equi sub. zooepidemicus is that it is a zoonotic pathogen, which use at industrial scale can create several risks. This creates opportunities for heterologous, or recombinant, production of HA. At an industrial scale, the recovery and purification of HA follow a series of precipitation and filtration steps. Current recombinant approaches are developing promising alternatives, although their industrial implementation has yet to be adequately assessed. The present study aims to create a theoretical framework to forecast the advantages and disadvantages of endogenous and recombinant strains in production with the same downstream strategy. The analyses included a selection of the best cost-related recombinant and endogenous production strategies, followed by a sensitivity analysis of different production variables in order to identify the three most critical parameters. Then, all variables were analyzed by varying them simultaneously and employing multiple linear regression. Results indicate that, regardless of HA source, production titer, recovery yield and bioreactor scale are the parameters that affect production costs the most. Current results indicate that recombinant production needs to improve current titer at least 2-fold in order to compete with costs of endogenous production. This study serves as a platform to inform decision-making for future developments and improvements in the recombinant production of HA.

Streptomyces are Gram-positive bacteria that occupy diverse ecological niches including host-associations with animals and plants. Members of this genus are known for their overwhelming repertoire of natural products, which has been exploited for almost a century as a source of medicines and agrochemicals. Notwithstanding intense scientific and commercial interest in Streptomyces natural products, surprisingly little is known of the intra- and/or inter-species ecological roles played by these metabolites. In this report we describe the chemical structures, biological properties, and biosynthetic relationships between natural products produced by Streptomyces isolated from internal tissues of predatory Conus snails, collected from the Great Barrier Reef, Australia. Using chromatographic, spectroscopic and bioassays methodology, we demonstrate that Streptomyces isolated from five different Conus species produce identical chemical and antifungal profiles – comprising a suite of polycyclic tetramic acid macrolactams (PTMs). To investigate possible ecological (and evolutionary) relationships we used genome analyses to reveal a close taxonomic relationship with other sponge-derived and free-living PTM producing Streptomyces (i.e., Streptomyces albus). In-depth phylogenomic analysis of PTM biosynthetic gene clusters indicated PTM structure diversity was governed by a small repertoire of genetic elements, including discrete gene acquisition events involving dehydrogenases. Overall, our study shows a Streptomyces-Conus ecological relationship that is concomitant with specific PTM chemical profiles. We provide an evolutionary framework to explain this relationship, driven by anti-fungal properties that protect Conus snails from fungal pathogens.