Genes that confer antibiotic resistance can rapidly be disseminated from one microorganism to another by mobile genetic elements, thus transferring resistance to previously susceptible bacterial strains. The misuse of antibiotics in health care and agriculture has provided a powerful evolutionary pressure to accelerate the spread of resistance genes, including those encoding β-lactamases. These are enzymes that are highly efficient in inactivating most of the commonly used β-lactam antibiotics. However, genes that confer antibiotic resistance are not only associated with pathogenic microorganisms, but are also found in non-pathogenic (i.e. environmental) microorganisms. Two recent examples are metal-dependent β-lactamases (MBLs) from the marine organisms
ROCker Models for Reliable Detection and Typing of Short-Read Sequences Carrying β-Lactamase Genes
ABSTRACT Identification of genes encoding β-lactamases (BLs) from short-read sequences remains challenging due to the high frequency of shared amino acid functional domains and motifs in proteins encoded by BL genes and related non-BL gene sequences. Divergent BL homologs can be frequently missed during similarity searches, which has important practical consequences for monitoring antibiotic resistance. To address this limitation, we built ROCker models that targeted broad classes (e.g., class A, B, C, and D) and individual families (e.g., TEM) of BLs and challenged them with mock 150-bp- and 250-bp-read data sets of known composition. ROCker identifies most-discriminant bit score thresholds in sliding windows along the sequence of the target protein sequence and hence can account for nondiscriminative domains shared by unrelated proteins. BL ROCker models showed a 0% false-positive rate (FPR), a 0% to 4% false-negative rate (FNR), and an up-to-50-fold-higher F1 score [2 × precision × recall/(precision + recall)] compared to alternative methods, such as similarity searches using BLASTx with various e-value thresholds and BL hidden Markov models, or tools like DeepARG, ShortBRED, and AMRFinder. The ROCker models and the underlying protein sequence reference data sets and phylogenetic trees for read placement are freely available through http://enve-omics.ce.gatech.edu/data/rocker-bla . Application of these BL ROCker models to metagenomics, metatranscriptomics, and high-throughput PCR gene amplicon data should facilitate the reliable detection and quantification of BL variants encoded by environmental or clinical isolates and microbiomes and more accurate assessment of the associated public health risk, compared to the current practice. IMPORTANCE Resistance genes encoding β-lactamases (BLs) confer resistance to the widely prescribed antibiotic class β-lactams. Therefore, it is important to assess the prevalence of BL genes in clinical or environmental samples for monitoring the spreading of these genes into pathogens and estimating public health risk. However, detecting BLs in short-read sequence data is technically challenging. Our ROCker model-based bioinformatics approach showcases the reliable detection and typing of BLs in complex data sets and thus contributes toward solving an important problem in antibiotic resistance surveillance. The ROCker models developed substantially expand the toolbox for monitoring antibiotic resistance in clinical or environmental settings.
more »
« less
- Award ID(s):
- 1759831
- NSF-PAR ID:
- 10354250
- Editor(s):
- Marshall, Christopher W.
- Date Published:
- Journal Name:
- mSystems
- Volume:
- 7
- Issue:
- 3
- ISSN:
- 2379-5077
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract Novosphingobium pentaromativorans andSimiduia agarivorans. Previous studies have demonstrated that their β-lactamase activity is comparable to those of well-known MBLs from pathogenic sources (e.g. NDM-1, AIM-1) but that they also possess efficient lactonase activity, an activity associated with quorum sensing. Here, we probed the structure and mechanism of these two enzymes using crystallographic, spectroscopic and fast kinetics techniques. Despite highly conserved active sites both enzymes demonstrate significant variations in their reaction mechanisms, highlighting both the extraordinary ability of MBLs to adapt to changing environmental conditions and the rather promiscuous acceptance of diverse substrates by these enzymes. -
null (Ed.)Staphylococci can cause a wide array of infections that can be life threatening. These infections become more deadly when the isolates are antibiotic resistant and thus harder to treat. Many resistance determinants are plasmid-mediated; however, staphylococcal plasmids have not yet been fully characterized. In particular, plasmids and their contributions to antibiotic resistance have not been investigated within the Arab states, where antibiotic use is not universally regulated. Here, we characterized the putative plasmid content among 56 Staphylococcus aureus and 10 Staphylococcus haemolyticus clinical isolates from Alexandria, Egypt. Putative plasmid sequences were detected in over half of our collection. In total, we identified 72 putative plasmid sequences in 27 S. aureus and 1 S. haemolyticus isolates. While these isolates typically carried one or two plasmids, we identified one isolate— S. aureus AA53—with 11 putative plasmids. The plasmid sequences most frequently encoded a Rep_1, RepL, or PriCT_1 type replication protein. As expected, antibiotic resistance genes were widespread among the identified plasmid sequences. Related plasmids were identified amongst our clinical isolates; homologous plasmids present in multiple isolates clustered into 11 groups based upon sequence similarity. Plasmids from the same cluster often shared antibiotic resistance genes, including blaZ , which is associated with β-lactam resistance. Our analyses suggest that plasmids are a key factor in the pathology and epidemiology of S. aureus in Egypt. A better characterization of plasmids and the role they contribute to the success of Staphylococci as pathogens will guide the design of effective control strategies to limit their spread.more » « less
-
null (Ed.)The horizonal transfer of plasmid-encoded genes allows bacteria to adapt to constantly shifting environmental pressures, bestowing functional advantages to their bacterial hosts such as antibiotic resistance, metal resistance, virulence factors, and polysaccharide utilization. However, common molecular methods such as short- and long-read sequencing of microbiomes cannot associate extrachromosomal plasmids with the genome of the host bacterium. Alternative methods to link plasmids to host bacteria are either laborious, expensive, or prone to contamination. Here we present the One-step Isolation and Lysis PCR (OIL-PCR) method, which molecularly links plasmid-encoded genes with the bacterial 16S rRNA gene via fusion PCR performed within an emulsion. After validating this method, we apply it to identify the bacterial hosts of three clinically relevant beta-lactamases within the gut microbiomes of neutropenic patients, as they are particularly vulnerable multidrug-resistant infections. We successfully detect the known association of a multi-drug resistant plasmid with Klebsiella pneumoniae , as well as the novel associations of two low-abundance genera, Romboutsia and Agathobacter . Further investigation with OIL-PCR confirmed that our detection of Romboutsia is due to its physical association with Klebsiella as opposed to directly harboring the beta-lactamase genes. Here we put forth a robust, accessible, and high-throughput platform for sensitively surveying the bacterial hosts of mobile genes, as well as detecting physical bacterial associations such as those occurring within biofilms and complex microbial communities.more » « less
-
null (Ed.)High-throughput sequencing is widely used for strain detection and characterization of antibiotic resistance in microbial metagenomic samples. Current analytical tools use curated antibiotic resistance gene (ARG) databases to classify individual sequencing reads or assembled contigs. However, identifying ARGs from raw read data can be time consuming (especially if assembly or alignment is required) and challenging, due to genome rearrangements and mutations. Here, we present the k-mer-based antibiotic gene resistance analyzer (KARGA), a multi-platform Java toolkit for identifying ARGs from metagenomic short read data. KARGA does not perform alignment; it uses an efficient double-lookup strategy, statistical filtering on false positives, and provides individual read classification as well as covering of the database resistome. On simulated data, KARGA’s antibiotic resistance class recall is 99.89% for error/mutation rates within 10%, and of 83.37% for error/mutation rates between 10% and 25%, while it is 99.92% on ARGs with rearrangements. On empirical data, KARGA provides higher hit score (≥1.5-fold) than AMRPlusPlus, DeepARG, and MetaMARC. KARGA has also faster runtimes than all other tools (2x faster than AMRPlusPlus, 7x than DeepARG, and over 100x than MetaMARC). KARGA is available under the MIT license at https://github.com/DataIntellSystLab/KARGA.more » « less
-
more » « less
Abstract Metallo-β-lactamases (MBLs) encoding carbapenem resistance in wastewater are a well-known serious threat to human health. Twelve Pseudomonas otitidis isolates obtained from a municipal wastewater treatment plant (WWTP) in Hawaii were found to possess a subclass B3 MBL – POM (P. otitidis MBL), with a minimum inhibition concentration (MIC) range of 8–16 mg/L. The unrooted neighbor-joining phylogenetic tree showed that these blaPOM genes isolated in wastewater samples (n = 12) were distinctly different from other reference genes isolated from clinical, freshwater, animal, and soil samples except for isolates MR7, MR8, and MR11. MR7, MR8, and MR11 were found to have 4, 3, and 3 amino acid substitutions when compared to the type strain MC10330T and were closely clustered to the clinical reference genes. The meropenem hydrolysis experiment showed that isolates with multiple amino acid substitutions completely hydrolyzed 64 mg/L of meropenem in 7 h. The emergence of the opportunistic pathogen P. otitidis chromosomally encoding blaPOM in the treated municipal wastewater is an alarming call for the spread of this MBL in the environment. Further studies are required to understand the mechanism and regulation of this carbapenem-resistant β-lactamase in order to fill in the knowledge gap.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/msystems.01281-21
