An overlooked phenomenon: complex interactions of potential error sources on the quality of bacterial de novo genome assemblies.

BACKGROUND: Parameters adversely affecting the contiguity and accuracy of the assemblies from Illumina next-generation sequencing (NGS) are well described. However, past studies generally focused on their additive effects, overlooking their potential interactions possibly exacerbating one another's effects in a multiplicative manner. To investigate whether or not they act interactively on de novo genome assembly quality, we simulated sequencing data for 13 bacterial reference genomes, with varying levels of error rate, sequencing depth, PCR and optical duplicate ratios. RESULTS: We assessed the quality of assemblies from the simulated sequencing data with a number of contiguity and accuracy metrics, which we used to quantify both additive and multiplicative effects of the four parameters. We found that the tested parameters are engaged in complex interactions, exerting multiplicative, rather than additive, effects on assembly quality. Also, the ratio of non-repeated regions and GC% of the original genomes can shape how the four parameters affect assembly quality. CONCLUSIONS: We provide a framework for consideration in future studies using de novo genome assembly of bacterial genomes, e.g. in choosing the optimal sequencing depth, balancing between its positive effect on contiguity and negative effect on accuracy due to its interaction with error rate. Furthermore, the properties of the genomes to be sequenced also should be taken into account, as they might influence the effects of error sources themselves.

Half-Sandwich Type Platinum-Group Metal Complexes of C-Glucosaminyl Azines: Synthesis and Antineoplastic and Antimicrobial Activities.

While platinum-based compounds such as cisplatin form the backbone of chemotherapy, the use of these compounds is limited by resistance and toxicity, driving the development of novel complexes with cytostatic properties. In this study, we synthesized a set of half-sandwich complexes of platinum-group metal ions (Ru(II), Os(II), Ir(III) and Rh(III)) with an N,N-bidentate ligand comprising a C-glucosaminyl group and a heterocycle, such as pyridine, pyridazine, pyrimidine, pyrazine or quinoline. The sugar-containing ligands themselves are unknown compounds and were obtained by nucleophilic additions of lithiated heterocycles to O-perbenzylated 2-nitro-glucal. Reduction of the adducts and, where necessary, subsequent protecting group manipulations furnished the above C-glucosaminyl heterocycles in their O-perbenzylated, O-perbenzoylated and O-unprotected forms. The derived complexes were tested on A2780 ovarian cancer cells. Pyridine, pyrazine and pyridazine-containing complexes proved to be cytostatic and cytotoxic on A2780 cells, while pyrimidine and quinoline derivatives were inactive. The best complexes contained pyridine as the heterocycle. The metal ion with polyhapto arene/arenyl moiety also impacted on the biological activity of the complexes. Ruthenium complexes with p-cymene and iridium complexes with Cp* had the best performance in ovarian cancer cells, followed by osmium complexes with p-cymene and rhodium complexes with Cp*. Finally, the chemical nature of the protective groups on the hydroxyl groups of the carbohydrate moiety were also key determinants of bioactivity; in particular, O-benzyl groups were superior to O-benzoyl groups. The IC50 values of the complexes were in the low micromolar range, and, importantly, the complexes were less active against primary, untransformed human dermal fibroblasts; however, the anticipated therapeutic window is narrow. The bioactive complexes exerted cytostasis on a set of carcinomas such as cell models of glioblastoma, as well as breast and pancreatic cancers. Furthermore, the same complexes exhibited bacteriostatic properties against multiresistant Gram-positive Staphylococcus aureus and Enterococcus clinical isolates in the low micromolar range.

Occurrence of Escherichia coli producing extended spectrum ß-lactamases in food-producing animals.

Multidrug resistance due to the production of extended-spectrum beta-lactamases (ESBLs) is a major problem in human as well as in veterinary medicine. These strains appear in animal and human microbiomes and can be the source of infection both in animal and in human healthcare, in accordance with the One Health theorem. In this study we examined the prevalence of ESBL-producing bacteria in food-producing animals. We collected 100 porcine and 114 poultry samples to examine the prevalence of ESBL producers. Isolates were identified using the MALDI-TOF system and their antibiotic susceptibility was tested using the disk diffusion method. ESBL gene families and phylogroups were detected by polymerase chain reactions. The prevalence of ESBL producers was relatively high in both sample groups: 72 (72.0%) porcine and 39 (34.2%) poultry isolates were ESBL producers. Escherichia coli isolates were chosen for further investigations. The most common ESBL gene was CTX-M-1 (79.3%). Most of the isolates belong to the commensal E. coli phylogroups. The porcine isolates could be divided into three phylogroups, while the distribution of the poultry isolates was more varied. In summary, ESBL-producing bacteria are prevalent in the faecal samples of the examined food-producing animals, with a dominance of the CTX-M-1 group enzymes and commensal E. coli phylogroups.

Consequences of rapid development owing to cohort splitting: just how costly is it to hurry?

In cohort splitting, diverging sub-cohorts may show substantial differences in their growth and developmental rates. Although in the past, causes and adaptive value of cohort splitting were studied in detail, individual-level consequences of cohort splitting are still rather overlooked. Life history theory predicts that considerably increased growth and developmental rates should be traded off against other costly life history traits. However, it is not clear whether one should expect such associations in adaptive developmental plasticity scenarios, because natural selection might have promoted genotypes that mitigate those potential costs of rapid development. To address these contrasting propositions, we assessed life history traits in the wolf spider Pardosa agrestis, both collected from natural habitat and reared in laboratory. We found that some traits are negatively associated with developmental rates in spiders collected from the wild, but these associations were relaxed to a considerable extent in laboratory-reared specimens. In general, we observed no consistent trend for the presence of developmental costs, although some results might suggest higher relative fecundity costs in rapidly developing females. Our study provides a detailed approach to the understanding of individual-level consequences of cohort splitting, and to the associations between key life history traits in adaptive developmental plasticity scenarios.

Candida biofilm production is associated with higher mortality in patients with candidaemia.

BACKGROUND: Candidaemia is a common life-threatening disease among hospitalised patients, but the effect of the Candida biofilm-forming ability on the clinical outcome remains controversial. OBJECTIVE: The aim was to determine the impact of biofilms, specifically focusing on biofilm mass and metabolic activity, on the mortality in candidaemia. PATIENTS/METHODS: The clinical data of patients (n = 127) treated at the University of Debrecen, Clinical Centre, between January 2013 and December 2018, were investigated retrospectively. Biofilm formation was assessed using the crystal violet and XTT assays, measuring the biofilm mass and metabolic activity, respectively. Isolates were classified as low, intermediate and high biofilm producers both regarding biofilm mass and metabolic activity. The susceptibility of one-day-old biofilms to fluconazole, amphotericin B, anidulafungin, caspofungin and micafungin was evaluated and compared to planktonic susceptibility. RESULTS: Intermediate/high biofilm mass was associated with significantly higher mortality (61%). All Candida tropicalis, Candida parapsilosis and Candida glabrata isolates originating from fatal infections were intermediate/high biofilm producers, whereas this ratio was 85% for Candida albicans. Solid malignancy was associated with intermediate/high biofilm producers (P = .043). The mortality was significantly higher in infections caused by Candida strains producing biofilms with intermediate/high metabolic activity (62% vs. 33%, P = .010). The ratio of concomitant bacteraemia was higher for isolates forming biofilms with low metabolic activity (53% vs 28%, P = .015). CONCLUSIONS: This study provides evidence that the Candida biofilms especially with intermediate/high metabolic activity are related to higher mortality in candidaemia.

Utilization of Vector Autoregressive and Linear Transfer Models to Follow Up the Antibiotic Resistance Spiral in Gram-negative Bacteria From Cephalosporin Consumption to Colistin Resistance.

BACKGROUND: Increasing antibiotic resistance may reciprocally affect consumption and lead to use of broader-spectrum alternatives; a vicious cycle that may gradually limit therapeutic options. Our aim in this study was to demonstrate this vicious cycle in gram-negative bacteria and show the utility of vector autoregressive (VAR) models for time-series analysis in explanatory and dependent roles simultaneously. METHODS: Monthly drug consumption data in defined daily doses per 100 bed-days and incidence densities of gram-negative bacteria (Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa, and Acinetobacter baumannii) resistant to cephalosporins or to carbapenems were analyzed using VAR models. These were compared to linear transfer models used earlier. RESULTS: In case of all gram-negative bacteria, cephalosporin consumption led to increasing cephalosporin resistance, which provoked carbapenem use and consequent carbapenem resistance and finally increased colistin consumption, exemplifying the vicious cycle. Different species were involved in different ways. For example, cephalosporin-resistant Klebsiella spp. provoked carbapenem use less than E. coli, and the association between carbapenem resistance of P. aeruginosa and colistin use was weaker than that of A. baumannii. Colistin use led to decreased carbapenem use and decreased carbapenem resistance of P. aeruginosa but not of A. baumannii. CONCLUSIONS: VAR models allow analysis of consumption and resistance series in a bidirectional manner. The reconstructed resistance spiral involved cephalosporin use augmenting cephalosporin resistance primarily in E. coli. This led to increased carbapenem use, provoking spread of carbapenem-resistant A. baumannii and consequent colistin use. Emergence of panresistance is fueled by such antibiotic-resistance spirals.

In vitro activity of rezafungin against common and rare Candida species and Saccharomyces cerevisiae.

BACKGROUND: Rezafungin is a novel echinocandin with excellent activity against common Candida species; however, limited data are available regarding rare Candida species. METHODS: We determined the in vitro susceptibility of 689 clinical isolates of 5 common and 19 rare Candida species, as well as Saccharomyces cerevisiae. The activity of rezafungin was compared with that of anidulafungin, caspofungin, micafungin, amphotericin B and fluconazole, using CLSI broth microdilution methodology (Fourth Edition: M27). RESULTS: Rezafungin MIC90 values were 0.06 mg/L for Candida albicans (n=125), Candida tropicalis (n=51), Candida dubliniensis (n=22), Candida inconspicua (n=41), Candida sojae (n=10), Candida lipolytica (n=10) and Candida pulcherrima (n=10), 0.12 mg/L for Candida glabrata (n=81), Candida krusei (n=53), Candida kefyr (n=52) and Candida fabianii (n=15), 0.25 mg/L for Candida lusitaniae (n=46) and Candida auris (n=19), 0.5 mg/L for Candida metapsilosis (n=15) and S. cerevisiae (n=21), 1 mg/L for Candida orthopsilosis (n=15) and Candida guilliermondii (n=27) and 2 mg/L for Candida parapsilosis sensu stricto (n=59). Caspofungin MIC90 values were 0.25-2 mg/L for all species, while micafungin and anidulafungin MIC90 values were similar to those of rezafungin. Fluconazole resistance was found in C. albicans (5.6%) and C. glabrata (4.9%); rezafungin was effective against these isolates as well. Amphotericin B MIC values did not exceed 2 mg/L. CONCLUSIONS: Rezafungin showed excellent in vitro activity against both WT and azole-resistant Candida species, as well as against S. cerevisiae. Rezafungin had similar activity to other echinocandins (excluding caspofungin) against common Candida species and, notably, against clinically relevant uncommon Candida species.

Physiological and Transcriptional Responses of Candida parapsilosis to Exogenous Tyrosol.

Tyrosol plays a key role in fungal morphogenesis and biofilm development. Also, it has a remarkable antifungal effect at supraphysiological concentrations. However, the background of the antifungal effect remains unknown, especially in the case of non-albicans Candida species such as Candida parapsilosis We examined the effect of tyrosol on growth, adhesion, redox homeostasis, virulence, as well as fluconazole susceptibility. To gain further insights into the physiological consequences of tyrosol treatment, we also determined genome-wide gene expression changes using transcriptome sequencing (RNA-Seq). A concentration of 15 mM tyrosol caused significant growth inhibition within 2 h of the addition of tyrosol, while the adhesion of yeast cells was not affected. Tyrosol increased the production of reactive oxygen species remarkably, as revealed by a dichlorofluorescein test, and it was associated with elevated superoxide dismutase, glutathione peroxidase, and catalase activities. The interaction between fluconazole and tyrosol was antagonistic. Tyrosol exposure resulted in 261 and 181 differentially expressed genes with at least a 1.5-fold increase or decrease in expression, respectively, which were selected for further study. Genes involved in ribosome biogenesis showed downregulation, while genes related to the oxidative stress response and ethanol fermentation were upregulated. In addition, tyrosol treatment upregulated the expression of efflux pump genes, including MDR1 and CDR1, and downregulated the expression of the FAD2 and FAD3 virulence genes involved in desaturated fatty acid formation. Our data demonstrate that exogenous tyrosol significantly affects the physiology and gene expression of C. parapsilosis, which could contribute to the development of treatments targeting quorum sensing in the future.IMPORTANCECandida-secreted quorum-sensing molecules (i.e., farnesol and tyrosol) are key regulators in fungal physiology, which induce phenotypic adaptations, including morphological changes, altered biofilm formation, and synchronized expression of virulence factors. Moreover, they have a remarkable antifungal activity at supraphysiological concentrations. Limited data are available concerning the tyrosol-induced molecular and physiological effects on non-albicans Candida species such as C. parapsilosis In addition, the background of the previously observed antifungal effect caused by tyrosol remains unknown. This study reveals that tyrosol exposure enhanced the oxidative stress response and the expression of efflux pump genes, while it inhibited growth and ribosome biogenesis as well as several virulence-related genes. Metabolism was changed toward glycolysis and ethanol fermentation. Furthermore, the initial adherence was not influenced significantly in the presence of tyrosol. Our results provide several potential explanations for the previously observed antifungal effect.

Comparison of Killing Activity of Micafungin Against Six Candida Species Isolated from Peritoneal and Pleural Cavities in RPMI-1640, 10 and 30% Serum.

Currently echinocandins are recommended in Candida peritonitis and pleuritis. We determined micafungin killing rates (k values) at therapeutic concentrations (0.25-2 mg/L) in RPMI-1640 with and without 10 and 30% serum mimicking in vivo conditions against six Candida species isolated from peritoneal and pleural fluid. In RPMI-1640, micafungin was fungicidal against C. glabrata, C. krusei and C. kefyr within 2.27 ± 10.68, 2.69 ± 10.29 and 3.10 ± 4.41 h, respectively, while was fungistatic against C. albicans, C. tropicalis and C. parapsilosis. In 10% serum, ≥ 0.25, ≥ 0.5, ≥ 0.5 and ≥ 1 mg/L micafungin produced positive k values (killing) for all C. albicans, C. glabrata, C. kefyr and C. krusei, respectively. In 30% serum, 2 mg/L micafungin produced killing against all C. albicans, C. glabrata and C. kefyr isolates, but was ineffective against C. krusei, C. parapsilosis and 2 of 3 C. tropicalis. Micafungin exposure should be increased against non-albicans species to eradicate fungi from peritoneal and pleural cavities.

Postantifungal effect of micafungin against Candida albicans, Candida dubliniensis and Candida africana in the presence and absence of serum.

We compared the micafungin killing rate and postantifungal effect (PAFE) at 4, 16 and 32 mg/L in RPMI- 1640 and in 50% serum against the C. albicans complex. In RPMI-1640 PAFEs were 1.5 - >19.4, 9.7 - >20.1 and 15.9 - >18.5 hours for C. albicans, C. africana and C. dubliniensis, respectively. In 50% serum PAFEs decreased sharply to 0-1.7 hours for all three species; killing rates were always negative. Short growth inhibition without killing in 50% serum suggests that micafungin PAFE has a limited role in the eradication of the C. albicans complex from the bloodstream.

Killing Activity of Micafungin Against Candida albicans, C. dubliniensis and Candida africana in the Presence of Human Serum.

We compared killing activity of micafungin in time-kill experiments in RPMI-1640 with and without 50% serum against Candida albicans, Candida dubliniensis and Candida africana reference strains and clinical isolates. Killing rates (k values) were determined for each strain and concentration. In RPMI-1640 MIC ranges were 0.015-0.03, 0.015-0.03 and 0.015 mg/L against C. albicans, C. dubliniensis and C. africana, respectively. In 50% serum MIC values for the three species increased 16- to 64-fold. In RPMI-1640 micafungin was fungicidal against two of three C. albicans isolates at 16 and 32 mg/L within 14.54 h and fungistatic against all C. africana and C. dubliniensis. Fifty per cent serum significantly decreased the growth rate of C. africana, but not of the other two species; weak in vivo replication ability of C. africana was confirmed in murine model. In 50% serum micafungin at 0.25 and 1 mg/L did not inhibit any of the three species (k values were always negative). Micafungin killing rate in 50% serum at 4, 16 and 32 mg/L was significantly decreased for C. albicans, but increased for C. dubliniensis compared to RPMI-1640. Killing activity of micafungin against C. africana was comparable or higher in 50% serum than in RPMI-1640. Although micafungin is a highly protein-bound drug, it was equally effective against the species of the C. albicans complex in 50% serum at therapeutic trough concentration (4 mg/L). Both in vitro and in vivo data confirmed the low virulence of C. africana compared to the two sibling species.

Comparison of rates of fecal colonization with extended-spectrum beta-lactamase-producing enterobacteria among patients in different wards, outpatients and medical students.

Because asymptomatic carriage of extended-spectrum beta-lactamase (ESBL) producers is a risk factor for infection, data on colonization dynamics are important when planning infection control. This study investigated fecal colonization with ESBL producers among inpatients, outpatients and medical students and compares the characteristics of ESBL producers among these groups. Carriage rates were investigated in 5581 fecal samples; 4343 from inpatients (330, 1397, 619 and 1864 from adult ICUs [intensive care units], adult non-ICUs, pediatric ICUs and pediatric non-ICUs, respectively), 814 from outpatients and 424 from screening of medical students. ESBL producers were characterized by co-resistance, integrons carried, and aminoglycoside resistance and ESBL genes. Dynamic regression models were built to identify relationships between combinations of time series of monthly antibiotic consumption, prevalence of carriers and infected subjects. Inpatients, ICU patients and adults showed higher prevalence than outpatients, non-ICU patients or children (7.4%, 9.3% and 12.0% vs. 3.1%, 6.1% and 4.1%, respectively). Klebsiella pneumoniae was more frequent in ICU patients; dominance of CTX-M-15 producers was more marked in adult than in pediatric inpatients. ESBL carriage was shown to be a consequence of infection in adults in the time-series analysis; antibiotic consumption had little effect. The epidemiology of colonization with ESBL producers differed between pediatric ICU, adult ICU and adult non-ICU patients. In adults, carriage of ESBL producers seems to be the consequence of infection, especially in ICU patients; the main source of colonization is nosocomial acquisition. In contrast, children are less likely to acquire colonizer strains in hospitals; importation of ESBL producers by colonized children seems to be significant.

Carriage rates and characteristics of Enterobacteriaceae producing extended-spectrum beta-lactamases in healthy individuals: comparison of applicants for long-term care and individuals screened for employment purposes.

AIMS: We compared the prevalence of extended-spectrum beta-lactamase (ESBL) producers in the faecal samples of 1,109 healthy individuals screened for employment purposes and in 531 asymptomatic individuals applying for long-term care (LTC). METHODS: Eosin-methylene blue agar plates supplemented with 2 mg/l cefotaxime were used to determine which individuals were ESBL producers. ESBL phenotype was confirmed by double-disk synergy test and ESBL genes were identified by sequencing. ESBL producers were characterized by co-resistance and integron carriage. RESULTS: ESBL producers were more frequent in the LTC applicants than in the employment screening individuals (7.2 vs. 2.0%; p < 0.0001), with 43 Escherichia coli, 18 Klebsiella pneumoniae, 1 Klebsiella oxytoca and 1 Proteus mirabilis being found. In the employment screening individuals, only E. coli was found. Most ESBL genes (79.4%, 50/63) were blaCTX-M type; blaCTX-M-15 was more frequent in the LTC applicants (p < 0.001). Regarding ESBL genes and integron diversity, E. coli isolates from the LTC applicants were more similar to K. pneumoniae than to E. coli from the employment screening individuals. CONCLUSION: These differences in the characteristics of ESBL producers may represent different sources of colonization. Most LTC applicants harboured K. pneumoniae or E. coli that were probably hospital-acquired whereas the E. coli isolates of many healthy individuals showed similarities to environmental E. coli.

Efficacy of single large doses of caspofungin in a neutropenic murine model against the "psilosis" group.

We compared the in vivo efficacy of single large dose of caspofungin to that of daily smaller caspofungin doses (with same cumulative doses) against C. albicans (echinocandin susceptible and resistant isolates) and the â€Å“psilosisâ€? group in a neutropenic murine model. Seven treatment groups were formed for C. orthopsilosis, C. metapsilosis and C. albicans (no treatment, 1, 2 and 3 mg/kg caspofungin daily for five days; single 5, 10 and 15 mg/kg caspofungin doses). For C. parapsilosis there were five treatment groups (no treatment, 3 and 4 mg/kg caspofungin daily for five days; single 15 and 20 mg/kg caspofungin). Tissue burdens of C. orthopsilosis and C. parapsilosis were significantly decreased by daily 3 mg/kg and 10 or 15 mg/kg single caspofungin doses (P<0.05-0.01) and daily 4 mg/kg and by single 15 and 20 mg/kg caspofungin doses (P<0.05-0.01), respectively. Against C. metapsilosis all treatment arms except the daily 1 mg/kg were effective (P<0.05-<0.001). Against C. albicans all treatment doses were effective. Neither daily 16 mg/kg nor single 80 mg/kg were effective against the resistant C. albicans strain. Higher doses and less frequent administration of caspofungin were comparable or sometimes superior to the lower, daily-dose regimen against the â€Å“psilosisâ€? group supporting further studies with this therapeutic strategy.

Killing rates for caspofungin against Candida albicans after brief and continuous caspofungin exposure in the presence and absence of serum.

It was previously demonstrated that brief (≤1 h) exposures to echinocandins are as effective to kill Candida albicans cells as continuous 24-h exposure. However, killing rates after continuous and short (1 h) echinocandin exposures to C. albicans have not yet been evaluated in RPMI-1640 with and without 50 % serum. We evaluated four echinocandin susceptible C. albicans bloodstream isolates, ATCC 10231 type strain and an echinocandin-resistant isolate (DPL20, FKS F645P). Caspofungin MICs, time-kill and postantifungal effect (PAFE) tests were performed in RPMI-1640 with and without 50 % serum. Killing rates (k values) in time-kill and PAFE experiments were determined for each strain and concentration. In time-kill experiments, colony count decreases were isolate- and concentration-dependent at 0.25, 1, 4, 8, 16 and 32 mg/L in RPMI-1640, but concentration-independent at 1, 4, 8, 16 and 32 mg/L in 50 % serum. One-hour caspofungin exposure at 4, 16 and 32 mg/L resulted in CFU decreases comparable with the results obtained in time-kill experiments in RPMI-1640, but 50 % serum at 4, 16 and 32 mg/L allowed growth of all isolates (k values were negative) (P < 0.05-0.001). PAFE in 50 % serum decreased markedly at 4, 16 and 32 mg/L. Killing rates remained high and concentration-independent in 50 % serum in case of continuous but not in case of brief caspofungin exposure. As only a short growth inhibition without killing was observed in 50 % serum, clinical relevance of caspofungin PAFE in vivo is questionable.

[Special application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry in clinical microbiological diagnostics]. / Mátrix-asszisztált lézer deszorpciós, ionizációs, repülési ido mérésén alapuló tömegspektrometria speciális alkalmazása a klinikai mikrobiológiai diagnosztika területén.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry as a new possibility for rapid identification of bacteria and fungi revolutionized the clinical microbiological diagnostics. It has an extreme importance in the routine microbiological laboratories, as identification of the pathogenic species rapidly will influence antibiotic selection before the final determination of antibiotic resistance of the isolate. The classical methods for identification of bacteria or fungi, based on biochemical tests, are influenced by many environmental factors. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry is a rapid method which is able to identify a great variety of the isolated bacteria and fungi based on the composition of conserved ribosomal proteins. Recently several other applications of the method have also been investigated such as direct identification of pathogens from the positive blood cultures. There are possibilities to identify bacteria from the urine samples in urinary tract infection or from other sterile body fluids. Using selective enrichment broth Salmonella sp from the stool samples can be identified more rapidly, too. The extended spectrum beta-lactamase or carbapenemase production of the isolated bacteria can be also detected by this method helping the antibiotic selection in some cases. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry based methods are suitable to investigate changes in deoxyribonucleic acid or ribonucleic acid, to carry out rapid antibiotic resistance determination or other proteomic analysis. The aim of this paper is to give an overview about present possibilities of using this technique in the clinical microbiological routine procedures.

Guideline for designing microbiome studies in neoplastic diseases.

Oncobiosis has emerged as a key contributor to the development, and modulator of the treatment efficacy of cancer. Hereby, we review the modalities through which the oncobiome can support the progression of tumors, and the emerging therapeutic opportunities they present. The review highlights the inherent challenges and limitations faced in sampling and accurately characterizing oncobiome. Additionally, the review underscores the critical need for the standardization of microbial analysis techniques and the consistent reporting of microbiome data. We provide a suggested metadata set that should accompany microbiome datasets from oncological settings so that studies remain comparable and decipherable.

Phylogeny of Transferable Oxazolidinone Resistance Genes and Homologs.

Oxazolidinone resistance, especially transmissible resistance, is a major public health concern, and the origin of this resistance mechanism is not yet resolved. This study aims to delve into the phylogenetic origin of the transmissible oxazolidinone resistance mechanisms conferring cross-resistance to other drugs of human and veterinary importance. The amino acid sequences of the five cfr ribosomal methylases and optrA and poxtA were used as queries in searches against 219,549 bacterial proteomes in the NCBI RefSeq database. Hits with >40% amino acid identity and >80% query coverage were aligned, and phylogenetic trees were reconstructed. All five cfr genes yielded highly similar trees, with rlmN housekeeping ribosomal methylases located basal to the sister groups of S-adenosyl-methionine-dependent methyltransferases from various Deltaproteobacteria and Actinomycetia, including antibiotic-producing Streptomyces species, and the monophyletic group of cfr genes. The basal branches of the latter contained paenibacilli and other soil bacteria; they then could be split into the clades [cfr(C):cfr(E)] and [[cfr:cfr(B)]:cfr(D)], always with different Bacillaceae in their stems. Lachnospiraceae were encountered in the basal branches of both optrA and poxtA trees. The ultimate origin of the cfr genes is the rlmN housekeeping ribosomal methylases, which evolved into a suicide-avoiding methylase in antibiotic producers; a soil organism (Lachnospiraceae, Paenibacilli) probably acted as a transfer organism into pathogenic bacteria. In the case of optrA, the porcine pathogenic Streptococcus suis was present in all branches, while the proteins closest to poxtA originated from Clostridia.

Monitoring Changes in the Antimicrobial-Resistance Gene Set (ARG) of Raw Milk and Dairy Products in a Cattle Farm, from Production to Consumption.

Raw milk and dairy products can serve as potential vectors for transmissible bacterial, viral and protozoal diseases, alongside harboring antimicrobial-resistance genes. This study monitors the changes in the antimicrobial-resistance gene pool in raw milk and cheese, from farm to consumer, utilizing next-generation sequencing. Five parallel sampling runs were conducted to assess the resistance gene pool, as well as phage or plasmid carriage and potential mobility. In terms of taxonomic composition, in raw milk the Firmicutes phylum made up 41%, while the Proteobacteria phylum accounted for 58%. In fresh cheese, this ratio shifted to 93% Firmicutes and 7% Proteobacteria. In matured cheese, the composition was 79% Firmicutes and 21% Proteobacteria. In total, 112 antimicrobial-resistance genes were identified. While a notable reduction in the resistance gene pool was observed in the freshly made raw cheese compared to the raw milk samples, a significant growth in the resistance gene pool occurred after one month of maturation, surpassing the initial gene frequency. Notably, the presence of extended-spectrum beta-lactamase (ESBL) genes, such as OXA-662 (100% coverage, 99.3% identity) and OXA-309 (97.1% coverage, 96.2% identity), raised concerns; these genes have a major public health relevance. In total, nineteen such genes belonging to nine gene families (ACT, CMY, EC, ORN, OXA, OXY, PLA, RAHN, TER) have been identified. The largest number of resistance genes were identified against fluoroquinolone drugs, which determined efflux pumps predominantly. Our findings underscore the importance of monitoring gene pool variations throughout the product pathway and the potential for horizontal gene transfer in raw products. We advocate the adoption of a new approach to food safety investigations, incorporating next-generation sequencing techniques.

Comprehensive Metagenomic Analysis of Veterinary Probiotics in Broiler Chickens.

Probiotics are widely used in broiler chickens to support the gut microbiome, gut health, and to reduce the amount of antibiotics used. Despite their benefits, there is concern over their ability to carry and spread antimicrobial resistance genes (ARGs), posing a significant public health risk. This study utilized next-generation sequencing to investigate ARGs in probiotics approved for poultry, focusing on their potential to be transferred via mobile genetic elements such as plasmids and phages. We examined the gut microbiome and resistome changes in 60 broiler chickens over their rearing period, correlating these changes with different probiotic treatments. Specific resistance mechanisms against critically important antibiotics were identified, including genes related to fluoroquinolone resistance and peptide antibiotic resistance. We also found genes with significant relevance to public health (aadK, AAC(6')-Ii) and multiple drug-resistance genes (vmlR, ykkC, ykkD, msrC, clbA, eatAv). Only one phage-encoded gene (dfrA43) was detected, with no evidence of plasmid or mobile genetic element transmission. Additionally, metagenomic analysis of fecal samples showed no significant changes corresponding to time or diet across groups. Our findings highlight the potential risks associated with the use of probiotics in poultry, particularly regarding the carriage of ARGs. It is crucial to conduct further research into the molecular genetics of probiotics to develop strategies that mitigate the risk of resistance gene transfer in agriculture, ensuring the safe and effective use of probiotics in animal husbandry.