In Vitro Ecological Response of the Human Gut Microbiome to Bioactive Extracts from Edible Wild Mushrooms.

This study presents the effect of two new products based on atomized extracts from edible wild mushrooms (RoBioMush1, RoBioMush2) on the microbiota of three target groups: clinically healthy (NG) individuals, individuals with nutritional disorders (ND), and individuals with cardiovascular diseases (CVD). The microbiota fingerprints were determined by quantitative polymerase chain reaction (qPCR). Modulations in the simulated microbiome were established and correlated with the presence of phenolic compounds released in the in vitro environment (a three-stage culture system GIS2 simulator, www.gissystems.ro). The high metabolizing capacity of NG and CVD correlated positively with the rest of the biological activities expressed in vitro. ND microbiota consumed a wide spectrum of monosaccharides from the products. Xylose was present in large quantities in the descending segment (minimum: 175 µg/mL for ND). The primary conclusion was that the microbiological ecosystem was modulated, as proven by the presence of specific biomarkers (e.g., ammonium levels and fingerprints of short-chain fatty acids⁻SCFAs), which stimulate the organism's health status and were correlated with the restoration of a normal microbiota fingerprint.

Phenotypic profiles of virulence in different Candida species isolated from vulvovaginal infections.

In order to establish an infection, pathogenic microorganisms have to colonize, survive, multiply, evade the immune system and spread to other tissues [1, 2, 3]. Although some Candida species are normally commensal in humans, in the last decades the frequency and the severity of nosocomial diseases due to Candida strains have increased dramatically [4]. The aim of the present study was to characterize some Candida strains isolated from vulvovaginal infections by determining the virulence and pathogenicity profile. The assessment of the in vitro expression of virulence cell wall associated factors (hyphal formation, adherence to HeLa cell line, biofilm development), soluble secreted enzymes (aspartyl protease, lipase, phospholipase, DN-ase) and Fe3+ accumulation was achieved by phenotypic methods on 13 yeast strains belonging to five Candida (C.) species (C. albicans, C. krusei, C. parapsilopsis, C. catenulata and C. kefyr). Candida sp. strains isolated from vulvovaginal infections showed species/ strain specific virulence profile.

In Vitro Modulatory Effect of Stevioside, as a Partial Sugar Replacer in Sweeteners, on Human Child Microbiota.

The effect of stevioside on human health is still insufficiently highlighted by recent research. The total or partial replacement of sugar with sweeteners influences the general state of health, especially the human microbiota's response as a determining factor in the onset of type 2 diabetes. The present study aimed to present the long-term (one-year) in vitro effect that regular stevioside consumption had on children's pattern microbiota. A metabolomic response was established by determining the synthesis of organic acids and a correlation with antioxidant status. An increase in the number of bacterial strains and the variation of amount of butyrate and propionate to the detriment of lactic acid was observed. The effect was evidenced by the progressive pH increasing, the reduction of acetic acid, and the proliferation of Escherichia coli strains during the simulations. Synthesis of the main short-chain fatty acids (SCFAs) was interpreted as a response (adaptation) of the microbiota to the stevioside, without a corresponding increase in antioxidant status. This study demonstrated the modulatory role of stevioside on the human microbiota and on the fermentation processes that determine the essential SCFA synthesis in maintaining homeostasis. The protection of the microbiota against oxidative stress was also an essential aspect of reducing microbial diversity.

Subtypes, resistance and virulence platforms in extended-drug resistant Acinetobacter baumannii Romanian isolates.

Acinetobacter baumannii has emerged worldwide as a dominant pathogen in a broad range of severe infections, raising an acute need for efficient antibacterials. This is the first report on the resistome and virulome of 33 extended drug-resistant and carbapenem-resistant A. baumannii (XDR CRAB) strains isolated from hospitalized and ambulatory patients in Bucharest, Romania. A total of 33 isolates were collected and analyzed using phenotypic antibiotic susceptibility and conjugation assays, PCR, whole-genome sequencing (WGS), pulsed-field gel electrophoresis (PFGE) and MultiLocus Sequence Typing (MLST). All isolates were extensively drug-resistant (XDR), being susceptible only to colistin. The carbapenem resistance was attributed by PCR mainly to blaOXA-24 and blaOXA-23 genes. PFGE followed by MLST analysis demonstrated the presence of nine pulsotypes and six sequence types. WGS of seven XDR CRAB isolates from healthcare-associated infections demonstrated the high diversity of resistance genes repertoire, as well as of mobile genetic elements, carrying ARGs for aminoglycosides, sulphonamides and macrolides. Our data will facilitate the understanding of resistance, virulence and transmission features of XDR AB isolates from Romanian patients and might be able to contribute to the implementation of appropriate infection control measures and to develop new molecules with innovative mechanisms of action, able to fight effectively against these bugs, for limiting the spread and decreasing the infection rate and mortality.

The Impact of Long-Term Antibiotic Therapy of Cutaneous Adverse Reactions to EGFR Inhibitors in Colorectal Cancer Patients.

Colorectal cancer (CRC) is an important public health issue, in terms of incidence and mortality, with approximately 1.8 million new cases reported worldwide in 2018. Advancements in understanding pathophysiological key steps in CRC tumorigenesis have led to the development of new targeted therapies such as those based on epidermal growth factor receptor inhibitors (EGFR inhibitors). The cutaneous adverse reactions induced by EGFR inhibitors, particularly papulopustular rash, often require long-term antibiotic treatment with tetracycline agents (mostly minocycline and doxycycline). However, this raises several issues of concern: possible occurrence of gut dysbiosis in already vulnerable CRC patients, selection of highly antibiotic resistant and/or virulent clones, development of adverse reactions related to tetracyclines, interference of antibiotics with the response to oncologic therapy, with a negative impact on disease prognosis etc. In the context of scarce information regarding these issues and controversial opinions regarding the role of tetracyclines in patients under EGFR inhibitors, our aim was to perform a thorough literature review and discuss the main challenges raised by long-term use of tetracyclines in advanced CRC patients receiving this targeted therapy.

The Bioactive Potential of Functional Products and Bioavailability of Phenolic Compounds.

The expression of bioactivity depends on the assimilation of different classes of natural substances (e.g., phenolic compounds) in vivo. Six functional extracts (Aspalathus linearis, leaves; Paullinia cupana, seeds; Aristotelia chilensis, berries; Ilex paraguariensis, leaves; Syzygium aromaticum, cloves, and wild berries) were analyzed in vitro and in vivo as an alternative to alleviating pathologies associated with oxidative stress (proliferation of cancer cells). The purpose of this research was to evaluate the in vitro and in vivo antioxidant and cytotoxic potential of hydroalcoholic solutions, in addition to the assimilation capacity of bioactive components in Saccharomyces boulardii cells. In vivo antioxidant capacity (critical point value) was correlated with the assimilation ratio of functional compounds. The results of in vitro antioxidant activities were correlated with the presence of quercetin (4.67 ± 0.27 mg/100 mL) and chlorogenic acid (14.38 ± 0.29 mg/100 mL) in I. paraguariensis. Bioassimilation of the main nutraceutical components depended on the individual sample. Phenolic acid levels revealed the poor assimilation of the main components, which could be associated with cell viability to oxidative stress.

Whole genome sequencing snapshot of multi-drug resistant Klebsiella pneumoniae strains from hospitals and receiving wastewater treatment plants in Southern Romania.

We report on the genomic characterization of 47 multi-drug resistant, carbapenem resistant and ESBL-producing K. pneumoniae isolates from the influent (I) and effluent (E) of three wastewater treatment plants (WWTPs) and from Romanian hospital units which are discharging the wastewater in the sampled WWTPs. The K. pneumoniae whole genome sequences were analyzed for antibiotic resistance genes (ARGs), virulence genes and sequence types (STs) in order to compare their distribution in C, I and E samples. Both clinical and environmental samples harbored prevalent and widely distributed ESBL genes, i.e. blaSHV, blaOXA, blaTEM and blaCTX M. The most prevalent carbapenemase genes were blaNDM-1, blaOXA-48 and blaKPC-2. They were found in all types of isolates, while blaOXA-162, a rare blaOXA-48 variant, was found exclusively in water samples. A higher diversity of carbapenemases genes was seen in wastewater isolates. The aminoglycoside modifying enzymes (AME) genes found in all types of samples were aac(6'), ant(2'')Ia, aph(3'), aaD, aac(3) and aph(6). Quinolone resistance gene qnrS1 and the multi-drug resistance oqxA/B pump gene were found in all samples, while qnrD and qnrB were associated to aquatic isolates. The antiseptics resistance gene qacEdelta1 was found in all samples, while qacE was detected exclusively in the clinical ones. Trimethroprim-sulfamethoxazole (dfrA, sul1 and sul2), tetracyclines (tetA and tetD) and fosfomycin (fosA6, known to be located on a transpozon) resistance genes were found in all samples, while for choramphenicol and macrolides some ARGs were detected in all samples (catA1 and catB3 / mphA), while other (catA2, cmIA5 and aac(6')Ib / mphE and msrE) only in wastewater samples. The rifampin resistance genes arr2 and 3 (both carried by class I integrons) were detected only in water samples. The highly prevalent ARGs preferentially associating with aquatic versus clinical samples could ascribe potential markers for the aquatic (blaSHV-145, qacEdelta1, sul1, aadA1, aadA2) and clinical (blaOXA-1, blaSHV-106,blaTEM-150, aac(3)Iia, dfrA14, oqxA10; oqxB17,catB3, tetD) reservoirs of AR. Moreover, some ARGs (oqxA10; blaSHV-145; blaSHV-100, aac(6')Il, aph(3')VI, armA, arr2, cmlA5, blaCMY-4, mphE, msrE, oqxB13, blaOXA-10) showing decreased prevalence in influent versus effluent wastewater samples could be used as markers for the efficiency of the WWTPs in eliminating AR bacteria and ARGs. The highest number of virulence genes (75) was recorded for the I samples, while for E and C samples it was reduced to half. The most prevalent belong to three functional groups: adherence (fim genes), iron acquisition (ent, fep, fyu, irp and ybt genes) and the secretion system (omp genes). However, none of the genes associated with hypervirulent K. pneumoniae have been found. A total of 14 STs were identified. The most prevalent clones were ST101, ST219 in clinical samples and ST258, ST395 in aquatic isolates. These STs were also the most frequently associated with integrons. ST45 and ST485 were exclusively associated with I samples, ST11, ST35, ST364 with E and ST1564 with C samples. The less frequent ST17 and ST307 aquatic isolates harbored blaOXA-162, which was co-expressed in our strains with blaCTX-M-15 and blaOXA-1.

Altered in Vitro Metabolomic Response of the Human Microbiota to Sweeteners.

Non-nutritive sweeteners represent an ingredient class that directly affects human health, via the development of inflammatory processes that promote chronic diseases related to microbiota dysbiosis. Several in vitro tests were conducted in the static GIS1 simulator. The aim of the study was to highlight the effect of sweeteners on the microbiota pattern of healthy individuals, associated with any alteration in the metabolomic response, through the production of organic acids and ammonium. The immediate effect of the in vitro treatment and the influence of the specific sweetener type on the occurrence of dysbiosis were evaluated by determining the biomarkers of the microbiota response. The presence of the steviol reduced the ammonium level (minimum of 410 mg/L), while the addition of cyclamate and saccharin caused a decrease in the number of microorganisms, in addition to lowering the total quantity of synthesized short-chain fatty acids (SCFAs). The bifidobacteria appeared to decrease below 102 genomes/mL in all the analyzed samples at the end of the in vitro simulation period. Barring the in vitro treatment of steviol, all the sweeteners tested exerted a negative influence on the fermentative profile, resulting in a decline in the fermentative processes, a rise in the colonic pH, and uniformity of the SCFA ratio.

An In Vitro Study of the Influence of Curcuma longa Extracts on the Microbiota Modulation Process, In Patients with Hypertension.

The multiple causes of cardiovascular diseases signify a major incidence and developmental risk of this pathology. One of the processes accountable for this pathologic development is the instauration of dysbiosis and its connection with an inflammatory process. Low antioxidant colonic protection encourages the progression of inflammation, with cardiovascular dysfunctions being a secondary consequence of the dysbiosis. Curcumin is one of the bioactive compounds displaying promising results for the reduction of an inflammatory process. The present study aims at demonstrating the capacity of three extracts drawn from Curcuma (C.) longa through an in vitro simulation process, for microbiota modulation in patients with hypertension. The acidic pH in the extraction process determined a high curcumin content in the extracts. The major phenolic compound identified was curcumin III, 622 ± 6.88 µg/mL for the ethanol/water/acetic acid extract. Low EC50 values were associated (0.2 µg/mL for DPPH scavenging activity) with the presence of curcumin isomers. A metabolic pattern became evident because the relationship between the short-chain fatty acids acted as a clinical biomarker. The curcumin present stimulated the formation of butyric and propionic acids. Microbiota activity control included a high degree of curcumin degradation and biotransformation in the other phenolic compounds. This developmental process was supported by the progression in the enterobacteria with a corresponding escalation in the pH level. The metabolomic pattern demonstrated a performance similar to the administration of dietary fibre, with the positive effects being dose-dependent.

In Vitro Human Microbiota Response to Exposure to Silver Nanoparticles Biosynthesized with Mushroom Extract.

The ability to orally administer silver nanoparticles (AgNPs) in enteric capsules implies a direct interaction with the colon microbiota. The in vitro effect provides a portrayal of the functional properties under in vivo conditions. The purpose of this study was to describe a green AgNP synthesis process, using aqueous extract from Lactarius piperatus mushroom, and to characterize the nanomaterial. We determined its antimicrobial and antioxidant effects in vitro in the microbiota of healthy individuals via the GIS1 system-a colon transit simulator. Per the quantitative polymerase chain reaction (qPCR) results, the antimicrobial properties of the AgNPs affected the initial share of different enteric species by decreasing the Bacteroides, Enterobacteriaceae, and Lactobacillus populations and favoring the Bifidobacterium group. The association between AgNPs and wild mushroom L. piperatus extract had a synergistic antibacterial activity against various pathogenic microorganisms while the mushroom extract reduced biofilm formation. Administration of AgNP maintained its constant antioxidant status, and it was correlated with a reduction in ammonium compounds. The physicochemical characterization of these NPs complemented their biochemical characterization. The maximum ultraviolet-visible spectroscopy (UV-VIS) absorbance was observed at 440 nm, while the Fourier transform infrared (FT-IR) spectrum reached a peak at 3296 cm⁻1, which was correlated with the high-performance liquid chromatographic analysis (HPLC). The major phenolic compound was homogentisic acid. The size (49 ± 16 nm in diameter) and spherical shape of the NPs were correlated with their biological effects in vitro.

Comparative Fingerprinting of the Human Microbiota in Diabetes and Cardiovascular Disease.

Diabetes and cardiovascular diseases are major causes of morbidity and mortality worldwide, and are associated with changes in the human gut microbiota. To better understand the relationships between diet, disease, and the colonic microbiome, we used the in vitro GIS1 system and repetitive element palindromic polymerase chain reaction (rep-PCR) to determine the microbial fingerprints in individuals with these diseases and compared them with the fingerprints in healthy controls. Clear differences were apparent in the three groups. The diabetes group showed significantly increased aerobic bacteria, increased coliforms, and reduced bifidobacteria; the balance between beneficial and pathogenic bacteria was disturbed; significant numbers of clostridia were present; and the proportions of various major bacterial groups were unstable through the length of the colon. The microbiota of the cardiovascular group had high numbers of beneficial strains and more closely resembled the control microbiota. Different patterns of lactic acid bacteria were observed in the three groups, and there was a direct link between the presence of lactate and the colonic pH. Ammonium, a microbial metabolite associated with colonic cancer, was associated with consistently high levels of Gram-positive bacteria in the diabetic patients. In the cardiovascular patients and controls, each colonic segment showed a distinct microbial fingerprint, whereas in the diabetics, the same rep-PCR profile occurred in all three segments. The diversity of beneficial bacteria was reduced in patients with a nutritional or cardiovascular disease. Both diabetes and cardiovascular disease are associated with changes in the colonic microbial fingerprint. This study of microbial microbiota fingerprint modification has direct applicability in medical practice.