The spread of extended-spectrum beta-lactamases (ESBLs) in nosocomial and community-acquired enterobacteria is an important challenge for clinicians due to the limited therapeutic options for ...infections that are caused by these organisms. Here, we developed a panel of ESBL coding genes, evaluated the abundance and prevalence of ESBL encoding genes in patients undergoing H. pylori eradication therapy, and summarized the effects of eradication therapy on functional profiles of the gut microbiome. To assess the repertoire of known beta lactamase (BL) genes, they were divided into clusters according to their evolutionary relation. Primers were designed for amplification of cluster marker regions, and the efficiency of this amplification panel was assessed in 120 fecal samples acquired from 60 patients undergoing H. pylori eradication therapy. In addition, fecal samples from an additional 30 patients were used to validate the detection efficiency of the developed ESBL panel. The presence for majority of targeted clusters was confirmed by NGS of amplification products. Metagenomic sequencing revealed that the abundance of ESBL genes within the pool of microorganisms was very low. The global relative abundances of the ESBL-coding gene clusters did not differ significantly among treatment states. However, at the level of each cluster, classical ESBL producers such as Klebsiella sp. for blaOXY (p = 0.0076), Acinetobacter sp. for blaADC (p = 0.02297) and others, differed significantly with a tendency to decrease compared to the pre- and post-eradication states. Only 13 clusters were common across all three datasets, suggesting a patient-specific distribution profile of ESBL-coding genes. The number of AMR genes detected in the post-eradication state was higher than that in the pre-eradication state, which could be attributed, at least in part, to the therapy. This study demonstrated that the ESBL screening panel was effective in targeting ESBL-coding gene clusters from bacterial DNA and that minor differences exist in the abundance and prevalence of ESBL-coding gene levels before and after eradication therapy.
It has been shown that the gut microbiota plays a central role in human health and disease. A wide range of volatile metabolites present in exhaled breath have been linked with gut microbiota and ...proposed as a non-invasive marker for monitoring pathological conditions. The aim of this study was to examine the possible correlation between volatile organic compounds (VOCs) in exhaled breath and the fecal microbiome by multivariate statistical analysis in gastric cancer patients (
= 16) and healthy controls (
= 33). Shotgun metagenomic sequencing was used to characterize the fecal microbiota. Breath-VOC profiles in the same participants were identified by an untargeted gas chromatography-mass spectrometry (GC-MS) technique. A multivariate statistical approach involving a canonical correlation analysis (CCA) and sparse principal component analysis identified the significant relationship between the breath VOCs and fecal microbiota. This relation was found to differ between gastric cancer patients and healthy controls. In 16 cancer cases, 14 distinct metabolites identified from the breath belonging to hydrocarbons, alcohols, aromatics, ketones, ethers, and organosulfur compounds were highly correlated with 33 fecal bacterial taxa (correlation of 0.891,
-value 0.045), whereas in 33 healthy controls, 7 volatile metabolites belonging to alcohols, aldehydes, esters, phenols, and benzamide derivatives correlated with 17 bacterial taxa (correlation of 0.871,
-value 0.0007). This study suggested that the correlation between fecal microbiota and breath VOCs was effective in identifying exhaled volatile metabolites and the functional effects of microbiome, thus helping to understand cancer-related changes and improving the survival and life expectancy in gastric cancer patients.
Objective Pseudomonas aeruginosa , a difficult-to-manage nosocomial pathogen, poses a serious threat to clinical outcomes in intensive care (ICU) patients due to its high antimicrobial resistance ...(AMR). To promote effective management, it is essential to investigate the genomic and phenotypic differences in AMR expression of the isolates. Methods A prospective observational study was conducted from July 2022 to April 2023 at Liepaja Regional Hospital in Latvia. The study included all adult patients who were admitted to the ICU and had a documented infection with P. aeruginosa , as confirmed by standard laboratory microbiological testing and short-read sequencing. Since ResFinder is the only sequencing-based database offering antibacterial susceptibility testing (AST) data for each antibiotic, we conducted a comparison of the resistance profile with the results of phenotypic testing, evaluating if ResFinder met the US Food and Drug Administration (FDA) requirements for approval as a new AMR diagnostic test. Next, to improve precision, AST data from ResFinder was compared with two other databases – AMRFinderPlus and RGI. Additionally, data was gathered from environmental samples to inform the implementation of appropriate infection control measures in real time. Results Our cohort consisted of 33 samples from 29 ICU patients and 34 environmental samples. The presence of P. aeruginosa infection was found to be associated with unfavourable clinical outcomes. A third of the patient samples were identified as multi-drug resistant isolates. Apart from resistance against colistin, significant discrepancies were observed when phenotypic data were compared to genotypic data. For example, the aminoglycoside resistance prediction of ResFinder yielded a major errors value of 3.03% for amikacin, which was marginally above the FDA threshold. Among the three positive environmental samples, one sample exhibited multiple AMR genes similar to the patient samples in its cluster. Conclusion Our findings underscore the importance of utilizing a combination of diagnostic methods for the identification of resistance mechanisms, clusters, and environmental reservoirs in ICUs.
Background
Helicobacter pylori (H. pylori) infection is the most extensively studied risk factor for gastric cancer. As with any bacteria, H. pylori will release distinctive odors that result from an ...emission of volatile metabolic byproducts in unique combinations and proportions. Effectively capturing and identifying these volatiles can pave the way for the development of innovative and non‐invasive diagnostic methods for determining infection. Here we characterize the H. pylori volatilomic signature, pinpoint potential biomarkers of its presence, and evaluate the variability of volatilomic signatures between different H. pylori isolates.
Materials and Methods
Using needle trap extraction, volatiles in the headspace above H. pylori cultures were collected and, following thermal desorption at 290°C in a splitless mode, were analyzed using gas chromatography–mass spectrometry. The resulting volatilomic signatures of H. pylori cultures were compared to those obtained from an analysis of the volatiles in the headspace above the cultivating medium only.
Results
Amongst the volatiles detected, 21 showed consistent differences between the bacteria cultures and the cultivation medium, with 11 compounds being elevated and 10 showing decreased levels in the culture's headspace. The 11 elevated volatiles are four ketones (2‐pentanone, 5‐methyl‐3‐heptanone, 2‐heptanone, and 2‐nonanone), three alcohols (2‐methyl‐1‐propanol, 3‐methyl‐1‐butanol, and 1 butanol), one aromatic (styrene), one aldehyde (2‐ethyl‐hexanal), one hydrocarbon (n‐octane), and one sulfur compound (dimethyl disulfide). The 10 volatiles with lower levels in the headspace of the cultures are four aldehydes (2‐methylpropanal, benzaldehyde, 3‐methylbutanal, and butanal), two heterocyclic compounds (2‐ethylfuran and 2‐pentylfuran), one ketone (2‐butanone), one aromatic (benzene), one alcohol (2‐butanol) and bromodichloromethane. Of the volatile species showing increased levels, the highest emissions are found to be for 3‐methyl‐1‐butanol, 1‐butanol and dimethyl disulfide. Qualitative variations in their emissions from the different isolates was observed.
Conclusions
The volatiles emitted by H. pylori provide a characteristic volatilome signature that has the potential of being developed as a tool for monitoring infections caused by this pathogen. Furthermore, using the volatilome signature, we are able to differentiate different isolates of H. pylori. However, the volatiles also represent potential confounders for the recognition of gastric cancer volatile markers.
Background
According to recent estimates 80% of Latvian population is infected with Helicobacter pylori thus their susceptibility to numerous gastric tract diseases is increased. The 1st line H. ...pylori eradication therapy includes treatment with clarithromycin in combination with amoxicillin or metronidazole and a proton pump inhibitor. However, potential adverse events caused by such therapies to microbiome are insufficiently studied.
Objective
This study aimed to evaluate the long‐term effect of H. pylori eradication on human gastrointestinal tract (GIT) microbiome.
Methods
The assessment of H pylori eradication impact on GIT microbiome was done by analyzing 120 samples acquired from 60 subjects. Each individual was prescribed the following 10‐day eradication regimen: Esomeprazolum 40 mg, Clarithromycinum 500 mg, and Amoxicillinum 1000 mg, BID. Samples from each individual were collected before starting H pylori eradication therapy, and 2 years after the completion of the therapy in OC‐Sensor (Eiken Chemical Co.) sample collection containers and stored at −86°C. Prior to DNA extraction, the samples were lyophilized, and total DNA was extracted using FastDNA Spin Kit for Soil. 16S V3 rRNA gene sequencing was done employing Ion Torrent PGM, and the obtained raw sequences were analyzed using vsearch and R (phyloseq, cluster packages).
Results
Alpha diversity measurements—observed OTUs, Chao1 and Shannon index did not differ significantly between the pre‐ and post‐eradication states (two‐tailed paired t test: P = .95; P = .71, P = .24, respectively). Unweighted and weighted UniFrac distances of beta diversity analysis indicated a non‐specific pattern of sample clustering. Enterotype shift was observed for the majority of individuals comparing pre‐ and post‐eradication study groups. Association analysis revealed that certain bacterial genera significantly correlated with age (eg, Dialister, Paraprevotella, Bifidobacterium), individual (eg, Thermotunica, Streptomyces, Faecalibacterium), and history of respiratory and/or allergic diseases (eg, Colinsella, Faecalibacterium). Redundancy analysis confirmed that the individual was a significant determinant of the subject's microbial community composition (ANOVA, 999 perm., P = .001) with the further lower impact of subject‐specific medical history (eg, medication used as prescribed: P = .005, history of cardiovascular diseases: P = .005, history of respiratory, and/or allergic diseases: P = .015) and physiological (eg, age: P = .005, gender: P = .02) parameters. In the post‐eradication study group, number of influential genera (n = 260) was increased compared to the pre‐eradication study group (n = 209).
Conclusion
Modest global differences at the community level exist between individuals before and after the eradication therapy; however, the microbiome structure is more related to the subject‐specific parameters rather than by the eradication therapy itself.
At the end of 2021, the SARS-CoV-2 Omicron variant of concern (VOC) displaced the previously dominant Delta VOC and enhanced diagnostic and therapeutic challenges worldwide. Respiratory specimens ...submitted to the Riga East University Hospital Laboratory Service by the central and regional hospitals of Latvia from January to March 2022 that were positive for SARS-CoV-2 RNA were tested by commercial multiplexed RT-qPCR targeting three of the Omicron VOC signature mutations: ΔH69/V70, E484A, and N501Y. Of the specimens tested and analyzed in parallel by whole-genome sequencing (WGS), 964 passed the internal quality criteria (genome coverage ≥90%, read depth ≥400×) and the Nextstrain’s quality threshold for “good”. We validated the detection accuracy of RT-qPCR for each target individually by using WGS as a control. The results were concordant with both approaches for 938 specimens, with the correct classification rate exceeding 96% for each target (CI 95%); however, the presumptive WHO label was misassigned for 21 specimens. The RT-qPCR genotyping provided an acceptable means to pre-monitor the prevalence of the two presumptive Omicron VOC sublineages, BA.1 and BA.2.
The Kluyveromyces marxianus yeast recently has gained considerable attention due to its applicability in high-value-added product manufacturing. In order to intensify the biosynthesis rate of a ...target product, reaching high biomass concentrations in the reaction medium is mandatory. Fed-batch processes are an attractive and efficient way how to achieve high cell densities. However, depending on the physiology of the particular microbial strain, an optimal media composition should be used to avoid by-product synthesis and, subsequently, a decrease in overall process effi-ciency. Thus, the aim of the present study was to optimise the synthetic growth medium and feeding solution compositions (in terms of carbon, nitrogen, phosphorous, magnesium, and calcium concentrations) for high cell density K. marxianus fed‑batch cultivations. Additionally, the biomass yields from the vitamin mixture and other macro/microelements were identified. A model predictive control algorithm was successfully applied for a fed-batch cultivation control. Biomass growth and substrate consumption kinetics were compared with the mathematical model predictions. Finally, 2‑phenylethanol biosynthesis was induced and its productivity was estimated. The determined optimal macronutrient ratio for K. marxianus biomass growth was identified as C:N:P = 1:0.07:0.011. The maximal attained yeast biomass concentration was close to 70 g·L-1 and the 2-PE biosynthesis rate was 0.372 g·L−1·h−1, with a yield of 74% from 2-phenylalanine.
Discrepancies between histology and serology results for
detection could be caused by a variety of factors, including a biopsy sampling error, expertise of the pathologist, natural loss of infection ...due to advanced atrophy, or a false-positive serology in the case of a previous infection, since antibodies may be present in blood following recovery from the infection.
To identify true
-positive individuals in discrepant cases by serology and histology using real time polymerase chain reaction (RT-PCR) as a gold standard.
Study subjects with discrepant histology and serology results were selected from the GISTAR pilot study data base in Latvia. Subjects having received previous
eradication therapy or reporting use of proton pump inhibitors, antibacterial medications, or bismuth containing drugs one month prior to upper endoscopy were excluded. We compared the discrepant cases to the corresponding results of RT-PCR performed on gastric biopsies.
In total, 97 individuals with discrepant results were identified: 81 subjects were serology-positive/histology-negative, while 16 were serology-negative/histology-positive. Among the serology-positive/histology-negative cases, 64/81 (79.0%) were false-positives by serology and, for the majority, inflammation was absent in all biopsies, while, in the serology-negative/histology-positive group, only 6.2% were proven false-positives by histology.
Among this high
prevalent, middle-aged population, the majority of discrepant cases between serology and histology were due to false positive-serology, rather than false-negative histology. This confirms the available evidence that the choice of treatment should not be based solely on the serological results, but also after excluding previous, self-reported eradication therapy.
The accuracy of plasma pepsinogen (Pg) as a marker for precancerous gastric lesions (PGL) has shown variable results. We aimed to identify factors associated with false negative (FN) cases in Pg ...testing and to adjust cut-off values for these factors in order to improve Pg yield. Plasma Pg was measured and upper endoscopy with biopsy was performed within the "Multicentric randomized study of
eradication and pepsinogen testing for prevention of gastric cancer mortality: the GISTAR study". A multivariable logistic model was built for FN and multiple factors. Values of Pg were compared and sensitivity and specificity were calculated using pre-existing Pg cut-offs for factors showing strong associations with FN. New cut-offs were calculated for factors that showed substantially lower sensitivity. Of 1210 participants, 364 (30.1%) had histologically confirmed PGL, of which 160 (44.0%) were FN. Current smokers, men, and
positives were more likely FN. Smoking in
negatives was associated with a higher Pg I/II ratio and substantially lower sensitivity of Pg testing than in other groups. Adjusting Pg cut-offs for current smokers by
presence improved sensitivity for detecting PGL in this group. Our study suggests that adjusting Pg cut-offs for current smokers by
status could improve Pg test performance.
Standard triple therapy is commonly prescribed Helicobacter pylori eradication regimen in Europe. However, the world is witnessing declines in eradication success. It is crucial to find better ...treatment options.
To evaluate efficacy, compliance and side effects of H. pylori eradication treatment by adding Saccharomyces boulardii .
We conducted a randomized clinical trial within the GISTAR cohort, consisting of healthy individuals aged 40-64 years. Participants were administered clarithromycin-containing triple therapy (clarithromycin 500 mg, amoxicillin 1000 mg, esomeprazole 40 mg) twice daily. Randomization was applied based on two factors: 1)addition of Saccharomyces boulardii CNCM I-745 500 mg BID or not; 2)treatment duration of 10 or 14 days. Treatment completion and adverse events were assessed via telephone interview 21-28 days after medication delivery. The efficacy was evaluated using a 13C-urea breath test (UBT) six months after treatment.
Altogether 404 participants were enrolled; data on adverse events were available from 391. Overall, 286 participants received follow-up UBT. Intention-to-treat analysis revealed higher eradication rates for 10-day probiotic treatment (70.8% vs. 54.6%, P = 0.022), but not for 14-day. Probiotic subgroups combined showed non-significantly higher efficacy in per-protocol analysis (90.6% vs. 85.0%, P = 0.183). S. boulardii reduced the frequency of adverse events ( P = 0.033) in 14-day regimen, particularly treatment-associated diarrhea ( P = 0.032). However, after the adjustment to control Type I error, results lost their significance.
Addition of S. boulardii to 14-day clarithromycin-containing triple regimen non-significantly lowers the likelihood of diarrhea and does not increase the eradication rate.
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