Gender assessment of the gut microbiome in obese patients

The aim. To assess the relationship between body mass index (BMI) and gut bacteria in men and women with obesity.Materials and methods. The study included 56 overweight patients, divided into 2 groups. The first group consisted of 27 women (the average age was 62 ± 2.2 years), the second group — 29 men (the average age was 55 ± 9 years). The Quetelet index (kg / m2) was calculated for all patients. To study the gut microbiome, the method of polymerase chain reaction in real time (RT-PCR) and metagenomic sequencing were used. DNA from feces was isolated using the Express-DNA-Bio DNA isolation kit (AlkorBio, Russia). To carry out RT-PCR, a set of reagents “Colonoflor-16” (“Alfalab”, Russia) was used. For microbiome sequencing, DNA libraries were prepared using the Illumina Nextera Sample Preparation Kit with DNA primers corresponding to the V3 — V4 regions of the 16S rRNA gene. The study of fecal samples was carried out using 16S rRNA gene sequencing on the Illumina platform (MiSeq sequencer).Results. It was revealed that a higher total number of bacteria, an increased content of Bacteroides fragilis group and Faeca-libacterium prausnitzii, is statistically significantly more common in women than in men. Strong negative correlations were found between BMI and total bacterial mass, between BMI and the number of Bacteroides fragilis group among women with grade I obesity. In overweight men, a correlation was found between BMI and the Bacteroides fragilis group / Faecalibacterium prausnitzii ratio.Conclusions. The total number of bacteria, the content of Bacteroides fragilis group and Faecalibacterium prausnitzii in the gut of patients have statistically significant associations with BMI, and probably can affect the formation of metabolic disorders to a greater extent in women than in men. To clarify the identified trends and patterns in this pilot study, further study of the microbiome with a large number of patients and additional analyzes of the metagenome (16S rRNA) and metabolome, a transcriptome, allowing to control the expression of key metabolic enzymes, largely associated with the compositional features of the gut microbiocenosis, is required.

ENDOTOXINEMIA IN PATIENTS WITH OBESITY AND NON-ALCOHOLIC FATTY LIVER DISEASE

Objective — to study the features of endotoxinemia and its relationship with changes in the intestinal microbial spectrum in obese patients with non-alcoholic fatty liver disease (NAFLD). Materials and methods. 84 patients with obesity and NAFLD were examined. The control group consisted of 20 apparently healthy individuals. The concentration of endotoxin (ET) in the blood serum was determined using the LAL Chromogenic Endpoint Assay kit manufactured by Hycult Biotech (Netherlands), relative quantitative composition of the microbiota at the level of basic phylotypes — by real-time polymerase chain reaction (CFX96Touch (Bio-Rad, USA)) using universal primers for the 16SpPHK gene and taxon-specific primers), quantitative composition of microbiota — using test of the «Colonoflor-16» system (Alfalab, RF) by real-time polymerase chain reaction.Results. Among patients with comorbidity of obesity and NAFLD, there were 40.48 % of males and 59.52 % of females, whose mean age was 53.72 ± 4.61 years. The ET level in the examined patients with comorbidity of obesity and NAFLC was significantly (p < 0.001) higher (1.01 ET/ml) than in the control group (0.60 ET/ml), was significantly (p < 0,05) is higher in women than in men (1.06 and 0.92 UE/ml, respectively) and increased with increasing age of patients (r = 0.30, p < 0.05). The ET level correlated with the relative content of Firmicutes (r = 0.39, p < 0.05) and their ratio in Bacteroidetes (r = 0.29, p < 0.05) and the level of Bifidobacterium spp. (r = 0 .37, p < 0 .05) a nd h ad a n i nverse r elationship w ith t he r elative c ontent o f B acteroidetes ( r = – 0.42, p < 0.01), including Bacteroides fragilis group (r = – 0.43, p < 0.01), Escherichia coli (r = – 0.41, p < 0.01) and total bacterial mass (r = – 0.39, p < 0.05). In the intestinal microbiota of the examined patients, a decrease in the representatives of the Lactobacillus spp. and Bifidobacterium spp. in 84.5 % and 30.9 % of patients, respectively, as well as Bacteroides thetaiotaomicron (88.0 %), Akkermansia muciniphila (79.8 %) and Faecalibacteriumprausnitzii (33.3 %). Whereas the number of gram-negative bacteria increased primarily due to Enterobacter spp, Citrobacter spp (45.2 %), Escherichia coli (19 %), Bacteroides fragilis group (29.8 %). It should be noted that the decrease in the level of A. muciniphila was often accompanied by an increase in the content of enterobacteria, while the excess content of B. fragilis group was accompanied by an increase above the upper limit of E. coli and the presence of anaerobic imbalance in the microbiota. In turn, increasing the number of Enterobacter spp. / Citrobacter spp. It was often accompanied by an increase in the content of E. coli and representatives of the B. fragilis group, against the background of a reduced number of A. muciniphila. Conclusions. The above indicates the presence of metabolic endotoxinemia in patients with comorbidity of obesity and NAFLD against the background of a decrease in the number of gram-positive anaerobic Lactobacillus spp. i Bifidobacterium spp. and bacteria involved in providing the intestinal barrier function (Bacteroides Thetaiotaomicron, Akkermansia muciniphila, Faecalibacterium prausnitzii), which creates conditions for an increased amount of ET into the blood and is especially important against the background of an increase in the content of gram-negative bacteria (representatives of gammaproteobacteria and bacteria of the Bacteroides fragilis), which are the source of ET.

Surveillance of Antimicrobial Susceptibility of Anaerobe Clinical Isolates in Southeast Austria: Bacteroides fragilis Group Is on the Fast Track to Resistance

Anaerobic bacteria play an important role in human infections. Bacteroides spp. are some of the 15 most common pathogens causing nosocomial infections. We present antimicrobial susceptibility testing (AST) results of 114 Gram-positive anaerobic isolates and 110 Bacteroides-fragilis-group-isolates (BFGI). Resistance profiles were determined by MIC gradient testing. Furthermore, we performed disk diffusion testing of BFGI and compared the results of the two methods. Within Gram-positive anaerobes, the highest resistance rates were found for clindamycin and moxifloxacin (21.9% and 16.7%, respectively), and resistance for beta-lactams and metronidazole was low (<1%). For BFGI, the highest resistance rates were also detected for clindamycin and moxifloxacin (50.9% and 36.4%, respectively). Resistance rates for piperacillin/tazobactam and amoxicillin/clavulanic acid were 10% and 7.3%, respectively. Two B. fragilis isolates were classified as multi-drug-resistant (MDR), with resistance against all tested beta-lactam antibiotics. The comparative study of 109 BFGI resulted in 130 discrepancies in 763 readings (17%) with a high number of Very Major Errors (VME) and Major Errors (ME). In summary, resistance rates, with the exception of clindamycin and moxifloxacin, are still low, but we are facing increasing resistance rates for BFGI. Surveillance studies on a regular basis are still recommended.

Antimicrobial Susceptibility of Bacteroides fragilis Group Isolates to Clindamycin, Tetracycline and Tigecycline, and Their Possession of tet and ermF Genes, which are Responsible for Resistance

Objective: We aimed to determine the resistance of Bacteroides fragilis group (BFG) bacteria to clindamycin, tetracycline and tigecycline and establish the distribution of related resistance genes. Method: In total 82 BFG strains, isolated from different clinical samples between January 2017 and December 2018, were identified by MALDI-TOF MS. Their antimicrobial sensitivities to were determined using agar dilution methodology (CLSI; M11-A7). The tetM, tetQ, tetX, tetX1, tet36, and ermF genes were investigated by PCR. Results: Eighty-two strains of BFG bacteria, isolated from intra-abdominal abscess (n=36), tissue biopsy (n=16), blood (n=14) and other sterile body fluids (n=12), were identified as Bacteroides fragilis (n=48), Bacteroides thetaiotaomicron (n=17), Bacteroides vulgatus (n=5), Bacteroides ovatus (n=4), Bacteroides caccae (n=1), Bacteroides uniformis (n=1) and Parabacteroides distasonis (n=6). The resistance rates to clindamycin, tetracycline and tigecycline were 54.9%, 84.1%, 4.9%, respectively. Non-B. fragilis isolates were more resistant than B. fragilis strains. In total, 57.3% of the isolates were ermF gene positive, while B. thetaiotaomicron had the highest rate (70.6%). The tet gene positivity ranged from 18.8% to 66.7% among species. The tetQ gene positivity was higher than other tet genes. The 92.7% of the isolates were resistant to at least one antibiotic, while 94% had at least one resistance gene. Conclusion: This study provided data on antimicrobial resistance of our BFG isolates to clindamycin, tetracycline and tigecycline and the related resistance genes. However, our information obtained could also be a starting point for further investigation of the antibiotic resistance mechanisms of Bacteroides species, as well as, resistance transfer among BFG isolates and other bacteria.

Comparison of the Antimicrobial Susceptibility Results of Bacteroides fragilis Group Isolates Determined by Agar Dilution and the Tentative EUCAST Disk Diffusion Method

Objective: In this study it was aimed to determine the antimicrobial susceptibility of Bacteroides fragilis group (BFG) bacteria using recently developed European Committee on Antimicrobial Susceptibility Testing (EUCAST) disc diffusion method and agar dilution method recommended by Clinical Laboratory Standart Institude (CLSI) for anaerobes and to investigate the agreement of the results of two tests. Method: The antimicrobial susceptibilities of a total of 56 BFG strains isolated from clinical samples and identified by MALDI-TOF MS analysis between January 2017 and December 2018, were tested to ampicillin, ampicillin/sulbactam, cefoxitin, imipenem, clindamycin, tigecycline, moxifloxacin and metronidazole MICs were determined by agar dilution method using sheep blood supplemented Brucella agar and disk diffusion test using host blood supplemented Fastidius Anaerobic Agar (FAA). Results: Six different BFG species consisting mostly strains of Bacteroides fragilis (n=34, 61%) and Bacteroides thetaiotaomicron (n=11, 20%) isolated from various clinical samples such as intraabdominal abscess (n= 24), blood (n=10) and tissue biopsy samples (n=11).were identified. Imipenem and metronidazole were the most effective antimicrobials with 98.2% susceptibility rates, followed by tigecycline, ampicillin/sulbactam, moxifloxacin and clindamycin with susceptibility rates of 89.3%, 66.1%, 57.1% and 46.4%, respectively. Most concordant results were obtained with metronidazole (100%), imipenem (89.8%) and tigecycline (89.8%). Acceptable compliance rates were not found for other antimicrobials. Conclusion: We can say that disc diffusion method is a fast, easy-to-apply, and reliable method used in clinical microbiology laboratories to determine the susceptibility of BFG bacteria to metronidazole, imipenem and tigecycline. However, to evolve a standard method especially for other antimicrobials, the experimental conditions should be optimized with studies dome with greater number of isolates.