Antimicrobial resistance is a major global health challenge. Metagenomics allows analyzing the presence and dynamics of "resistomes" (the ensemble of genes encoding antimicrobial resistance in a ...given microbiome) in disparate microbial ecosystems. However, the low sensitivity and specificity of available metagenomic methods preclude the detection of minority populations (often present below their detection threshold) and/or the identification of allelic variants that differ in the resulting phenotype. Here, we describe a novel strategy that combines targeted metagenomics using last generation in-solution capture platforms, with novel bioinformatics tools to establish a standardized framework that allows both quantitative and qualitative analyses of resistomes.
We developed ResCap, a targeted sequence capture platform based on SeqCapEZ (NimbleGene) technology, which includes probes for 8667 canonical resistance genes (7963 antibiotic resistance genes and 704 genes conferring resistance to metals or biocides), and 2517 relaxase genes (plasmid markers) and 78,600 genes homologous to the previous identified targets (47,806 for antibiotics and 30,794 for biocides or metals). Its performance was compared with metagenomic shotgun sequencing (MSS) for 17 fecal samples (9 humans, 8 swine). ResCap significantly improves MSS to detect "gene abundance" (from 2.0 to 83.2%) and "gene diversity" (26 versus 14.9 genes unequivocally detected per sample per million of reads; the number of reads unequivocally mapped increasing up to 300-fold by using ResCap), which were calculated using novel bioinformatic tools. ResCap also facilitated the analysis of novel genes potentially involved in the resistance to antibiotics, metals, biocides, or any combination thereof.
ResCap, the first targeted sequence capture, specifically developed to analyze resistomes, greatly enhances the sensitivity and specificity of available metagenomic methods and offers the possibility to analyze genes related to the selection and transfer of antimicrobial resistance (biocides, heavy metals, plasmids). The model opens the possibility to study other complex microbial systems in which minority populations play a relevant role.
The carriage of two important pathogens of pigs, that is enterotoxigenic Escherichia coli (ETEC) and Clostridioides difficile, was investigated in 104 cloacal samples from wild griffon vultures (Gyps ...fulvus) fed on pig carcasses at supplementary feeding stations (SFS), along with their level of antimicrobial resistance (AMR). E. coli was isolated from 90 (86.5%) samples, but no ETEC was detected, likely because ETEC fimbriae confer the species specificity of the pathogen. Resistance to at least one antimicrobial agent was detected in 89.9% of E. coli isolates, with AMR levels being extremely high (>70%) for tetracycline and streptomycin and very high (>50%) for ampicillin and sulfamethoxazole–trimethoprim. Resistance to other critically important antimicrobials such as colistin and extended‐spectrum cephalosporins was 2.2% and 1.1%, respectively, and was encoded by the mcr‐1 and blaSHV‐12 genes. Multidrug resistance was displayed by 80% of the resistant E. coli, and blaSHV‐12 gene shared plasmid with other AMR genes. In general, resistance patterns in E. coli from vultures mirrored those found in pigs. Clostridioides difficile was detected in three samples (2.9%); two of them belonged to PCR ribotype 078 and one to PCR ribotype 126, both commonly found in pigs. All C. difficile isolates were characterized by a moderate‐to‐high level of resistance to fluoroquinolones and macrolides but susceptible to metronidazole or vancomycin, similar to what is usually found in C. difficile isolates from pigs. Thus, vultures may contribute somewhat to the environmental dissemination of some pig pathogens through their acquisition from pig carcasses and, more importantly, of AMR for antibiotics of critical importance for humans. However, the role of vultures would likely be much lesser than that of disposing pig carcasses at the SFS. The monitoring of AMR, and particularly of colistin‐resistant and ESBL‐producing E. coli, should be considered in pig farms used as sources of carcasses for SFS.
Metagenomic sequencing is a promising method that has the potential to revolutionize the world of pathogen detection and antimicrobial resistance (AMR) surveillance in food-producing environments. ...However, the analysis of the huge amount of data obtained requires performant bioinformatics tools and databases, with intuitive and straightforward interpretation. In this study, based on long-read metagenomics data of chicken fecal samples with a spike-in mock community, we proposed confidence levels for taxonomic identification and AMR gene detection, with interpretation guidelines, to help with the analysis of the output data generated by KMA, a popular
mer read alignment tool. Additionally, we demonstrated that the completeness and diversity of the genomes present in the reference databases are key parameters for accurate and easy interpretation of the sequencing data. Finally, we explored whether KMA, in a two-step procedure, can be used to link the detected AMR genes to their bacterial host chromosome, both detected within the same long-reads. The confidence levels were successfully tested on 28 metagenomics datasets which were obtained with sequencing of real and spiked samples from fecal (chicken, pig, and buffalo) or food (minced beef and food enzyme products) origin. The methodology proposed in this study will facilitate the analysis of metagenomics sequencing datasets for KMA users. Ultimately, this will contribute to improvements in the rapid diagnosis and surveillance of pathogens and AMR genes in food-producing environments, as prioritized by the EU.
The study assessed the impact of four equine semen processing techniques on sperm quality and microbial load immediately post-processing and after 48 h of refrigeration. The aim was to explore the ...potential reduction of prophylactic antibiotic usage in semen extenders. Semen from ten adult stallions was collected and processed under a strict hygiene protocol and divided into four aliquots: Simple Centrifugation with antibiotics (SC+), Simple Centrifugation (SC-), Single-Layer Colloidal Centrifugation (CC-), and Filtration (with SpermFilter
) (F-), all in extenders without antibiotics. Sperm motility, viability, and microbial load on three culture media were assessed. No significant differences were observed in the main in the sperm quality parameters among the four protocols post-processing and at 48 h (
< 0.05 or
< 0.1). Microbial loads in Columbia 5% Sheep Blood Agar and Schaedler vitamin K1 5% Sheep Blood Agar mediums were significantly higher (
< 0.10) for raw semen than for CS+, CC-, and F- post-processing. For Sabouraud Dextrose Agar medium, the microbial load was significantly higher (
< 0.10) in raw semen compared to CS+ and F-. No significant differences (
< 0.10) were found in 48 h chilled samples. Regardless of antibiotic presence, the evaluated processing methods, when combined with rigorous hygiene measures, maintained semen quality and reduced microbial load to the same extent as a traditional protocol using antibiotics.
Many articles have been published on resistant microorganisms isolated from humans, animals, foods and the environment in Ghana. However, there are no reviews that summarize the information on the ...isolates and antibiotics tested so far in the country. This literature review was completed through "PubMed" and "Google Scholar" searches. We included publications from the period 1975-2015 with a laboratory-based methodology to determine antibiotic resistance of strains isolated in Ghana. In total, 60 articles were included in the analysis with 10% of the articles carrying out nationwide research on antibiotic resistance. The regions of Ghana with the highest published articles were Greater Accra (40%), Ashanti (21.7%) and Northern Region (10%). Most of the studies (86.7%) were related to isolates collected from human samples followed by environmental (5%), animal (3%) and food samples (2%). Ten different bacteria genera were observed in the studies. The most common was
, followed by
spp.,
spp. and
spp. The highest mean resistance rate was encountered in
(62.2%) followed by
spp
(60.4%) and
spp. (52.1%). High resistance rates have been found in Ghana, however, the data are skewed and some regions of the country have been neglected. There is a need for higher quality research to establish and monitor resistance patterns in Upper West, Brong-Ahafo, Volta and Eastern Regions of Ghana.
Plasmid-mediated antimicrobial resistance is one of the major threats to public health worldwide. The mechanisms involved in the plasmid/host coadaptation are still poorly characterized, and their ...understanding is crucial to comprehend the genesis and evolution of multidrug-resistant bacteria. With this purpose, we designed an experimental evolution using Haemophilus influenzae RdKW20 as the model strain carrying the ColE1-like plasmid pB1000. Five H. influenzae populations adapted previously to the culture conditions were transformed with pB1000 and subsequently evolved to compensate for the plasmid-associated fitness cost. Afterward, we performed an integrative multiomic analysis combining genomics, transcriptomics, and metabolomics to explore the molecular mechanisms involved in the compensatory evolution of the plasmid. Our results demonstrate that minimal modifications in the host are responsible for plasmid adaptation. Among all of them, the most enriched process was amino acid metabolism, especially those pathways related to serine, tryptophan, and arginine, eventually related to the genesis and resolution of plasmid dimers. Additional rearrangements occurred during the plasmid adaptation, such as an overexpression of the ribonucleotide reductases and metabolic modifications within specific membrane phospholipids. All these findings demonstrate that the plasmid compensation occurs through the combination of diverse host-mediated mechanisms, of which some are beyond genomic and transcriptomic modifications.
The ability of bacteria to horizontally transfer genetic material has turned antimicrobial resistance into one of the major sanitary crises of the 21st century. Plasmid conjugation is considered the main mechanism responsible for the mobilization of resistance genes, and its understanding is crucial to tackle this crisis. It is generally accepted that the acquisition and maintenance of mobile genetic elements entail a fitness cost to its host, which is susceptible to be alleviated through a coadaptation process or compensatory evolution. Notwithstanding, despite recent major efforts, the underlying mechanisms involved in this adaptation remain poorly characterized. Analyzing the plasmid/host coadaptation from a multiomic perspective sheds light on the physiological processes involved in the compensation, providing a new understanding on the genesis and evolution of plasmid-mediated antimicrobial-resistant bacteria.
Haemophilus parasuis, the causative agent of Glässer's disease, is one of the early colonizers of the nasal mucosa of piglets. It is prevalent in swine herds, and lesions associated with disease are ...fibrinous polyserositis and bronchopneumonia. Antibiotics are commonly used in disease control, and resistance to several antibiotics has been described in H. parasuis. Prediction of H. parasuis virulence is currently limited by our scarce understanding of its pathogenicity. Some genes have been associated with H. parasuis virulence, such as lsgB and group 1 vtaA, while biofilm growth has been associated with nonvirulent strains. In this study, 86 H. parasuis nasal isolates from farms that had not had a case of disease for more than 10 years were obtained by sampling piglets at weaning. Isolates were studied by enterobacterial repetitive intergenic consensus PCR and determination of the presence of lsgB and group 1 vtaA, biofilm formation, inflammatory cell response, and resistance to antibiotics. As part of the diversity encountered, a novel 2,661-bp plasmid, named pJMA-1, bearing the blaROB-1 β-lactamase was detected in eight colonizing strains. pJMA-1 was shown to share a backbone with other small plasmids described in the Pasteurellaceae, to be 100% stable, and to have a lower biological cost than the previously described plasmid pB1000. pJMA-1 was also found in nine H. parasuis nasal strains from a separate collection, but it was not detected in isolates from the lesions of animals with Glässer's disease or in nontypeable Haemophilus influenzae isolates. Altogether, we show that commensal H. parasuis isolates represent a reservoir of β-lactam resistance genes which can be transferred to pathogens or other bacteria.
Antimicrobial resistance is a major health problem. After decades of research, numerous difficulties in tackling resistance have emerged, from the paucity of new antimicrobials to the inefficient ...contingency plans to reduce the use of antimicrobials; consequently, resistance to these drugs is out of control. Today we know that bacteria from the environment are often at the very origin of the acquired resistance determinants found in hospitals worldwide. Here we define the genetic components that flow from the environment to pathogenic bacteria and thereby confer a quantum increase in resistance levels, as resistance units (RU). Environmental bacteria as well as microbiomes from humans, animals, and food represent an infinite reservoir of RU, which are based on genes that have had, or not, a resistance function in their original bacterial hosts. This brief review presents our current knowledge of antimicrobial resistance and its consequences, with special focus on the importance of an ecologic perspective of antimicrobial resistance. This discipline encompasses the study of the relationships of entities and events in the framework of curing and preventing disease, a definition that takes into account both microbial ecology and antimicrobial resistance. Understanding the flux of RU throughout the diverse ecosystems is crucial to assess, prevent and eventually predict emerging scaffolds before they colonize health institutions. Collaborative horizontal research scenarios should be envisaged and involve all actors working with humans, animals, food and the environment.
Highlights • Genomic and phenotypic characterisation of colistin-resistant Acinetobacter baumannii strains was performed. • Two mutations in pmrB caused colistin resistance in separate A. baumannii ...isolates. • The P233S mutation could lead to an altered virulence profile. • The novel ΔI19 mutation does not appear to affect in vitro virulence. • blaGES-5 was identified in a set of isolates for the first time in A. baumannii.
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