Comprehensive Antibiotic Resistance Database Research Articles

Enriching the future of public health microbiology with hybridization bait capture.

SUMMARYPublic health microbiology focuses on microorganisms and infectious agents that impact human health. For years, this field has relied on culture or molecular methods to investigate complex samples of public health importance. However, with the increase in accuracy and decrease in sequencing cost over the last decade, there has been a transition to the use of next-generation sequencing in public health microbiology. Nevertheless, many available sequencing methods (e.g., shotgun metagenomics and amplicon sequencing) do not work well in complex sample types, require deep sequencing, or have inherent biases associated with them. Hybridization bait capture, also known as target enrichment, brings in solutions for such limitations. It is an increasingly popular technique to simultaneously characterize many thousands of genetic elements while reducing the amount of sequencing needed (thereby reducing the sequencing costs). Here, we summarize the concept of hybridization bait capture for public health, reviewing a total of 35 bait sets designed in six key topic areas for public health microbiology [i.e., antimicrobial resistance (AMR), bacteria, fungi, parasites, vectors, and viruses], and compare hybridization bait capture to previously relied upon methods. Furthermore, we provide an in-depth comparison of the three most popular bait sets designed for AMR by evaluating each of them against three major AMR databases: Comprehensive Antibiotic Resistance Database, Microbial Ecology Group Antimicrobial Resistance Database, and Pathogenicity Island Database. Thus, this article provides a review of hybridization bait capture for public health microbiologists.

Whole genome sequencing and antimicrobial resistance among clinical isolates of Shigella sonnei in Addis Ababa, Ethiopia.

Shigellosis is an acute gastroenteritis infection and one of Ethiopia's most common causes of morbidity and mortality, especially in children under five. Antimicrobial resistance (AMR) has spread quickly among Shigella species due to inappropriate antibiotic use, inadequacies of diagnostic facilities, and unhygienic conditions. This study aimed to characterize Shigella sonnei (S. sonnei) using whole genome sequence (WGS) analysis in Addis Ababa, Ethiopia. The raw reads were quality-filtered and trimmed, and a minimum length of 50bp was retained and taxonomically classified using MiniKraken version 1. The whole genome data were aligned with Antibiotic Resistance Gene (ARG) sequences of the Comprehensive Antibiotic Resistance Database (CARD) by Resistance Gene Identifier (RGI). Plasmids were analyzed using the PlasmidFinder tool version 2.1. Additionally, AMR and virulence genes were screened at the Centre for Genomic Epidemiology (CGE) web-based server. All isolates in our investigation contained genes encoding blaEC-8 and blaZEG-1. Here, 60.7% of the isolates were phenotypically sensitive to cefoxitin among the blaEC-8 genes detected in the genotyping analysis, whereas all isolates were completely resistant to amoxicillin and erythromycin phenotypically. The study also identified genes that conferred resistance to trimethoprim (dfrA). Plasmid Col156 and Col (BS512) types were found in all isolates, while IncFII and Col (MG828) plasmids were only identified in one isolate. This study found that many resistant genes were present, confirming the high variety in S. sonnei strains and hence a divergence in phylogenetic relationships. Thus, combining WGS methods for AMR prediction and strain identification into active surveillance may be beneficial for monitoring the spread of AMR in S. sonnei and detecting the potential emergence of novel variations.

Genomic characterization of Pantoea dispersa A003 isolated from a clinical patient

IntroductionPantoea dispersa is a Gram-negative bacterium generally considered as a plant pathogen and rarely causes human infections. To date, there have been 13 studies that have documented clinical infections linked to P. dispersa, with a primary emphasis on the initial identification of this pathogen. The genomic features and the mechanisms underlying the pathogenesis of P. dispersa remain largely uninvestigated. In the present study, we describe a clinical infection caused by P. dispersa and provide the first genomic analysis of this bacterium.MethodsThe bacterial strain designated A003 was isolated from blood samples. Preliminary identification of strain A003 was conducted using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) technology (VITEK® MS, bioMérieux, France). Biochemical and antimicrobial susceptibility assessments were carried out utilizing the VITEK-2 compact automated microbial analysis system (bioMérieux, France). Additionally, whole genome sequencing and subsequent analysis were performed to further elucidate the potential pathogenicity.ResultsThe bacterial isolate was cultured overnight at 35°C in a CO2-enriched environment on Columbia blood agar, resulting in the appearance of a white, smooth, Gram-negative rod-shaped bacterium with diameters ranging from 1 to 2 mm. Identification of strain A003 was achieved with a high confidence level of 99.9% as P. dispersa using MALDI-TOF mass spectrometry. The average nucleotide identity (ANI) value between strain A003 and the reference strain P. dispersa DSM 30073T was 98.08%, while the DNA–DNA hybridization (DDH) value was 84.10% (Formula 2) based on genome sequencing data, which further confirmed that A003 belonged to the P. dispersa species. Comprehensive analysis revealed the presence of 372 virulence factors, 15 antibiotic resistance genes, 222 carbohydrate-active enzymes, and 666 genes related to Type III secretion system (T3SS) effector proteins, as identified through the core databases of VFDB (Virulence Factors of Pathogenic Bacteria), CARD (Comprehensive Antibiotic Resistance Database), CAZY (Carbohydrate-Active enZYmes Database), and T3SS. The virulence factors included type IV pili (TFP), type VI secretion system, flagella, iron uptake system, and ompA, which are implicated in bacterial pathogenicity. The antibiotic resistance profile indicated resistance to fluoroquinolones, cephalosporins, penams, macrolides, and aminoglycosides, as annotated by the CARD database.ConclusionThe draft genome sequenced represents the inaugural genomic sequence of P. dispersa derived from clinical sources. This advancement may enhance clinical practitioners’ comprehension of the organism’s clinical attributes and serve as a foundational resource for future research into its virulence, antibiotic resistance, and host–pathogen interactions.

Detection of Antimicrobial Proteins/Peptides and Bacterial Proteins Involved in Antimicrobial Resistance in Raw Cow's Milk from Different Breeds.

Proteins involved in antibiotic resistance (resistome) and with antimicrobial activity are present in biological specimens. This study aims to explore the presence and abundance of antimicrobial peptides (AMPs) and resistome proteins in bovine milk from diverse breeds and from intensive (Pezzata rossa, Bruna alpina, and Frisona) and non-intensive farming (Podolica breeds). Liquid atmospheric pressure matrix-assisted laser desorption/ionization (LAP-MALDI) mass spectrometry (MS) profiling, bottom-up proteomics, and metaproteomics were used to comprehensively analyze milk samples from various bovine breeds in order to identify and characterize AMPs and to investigate resistome proteins. LAP-MALDI MS coupled with linear discriminant analysis (LDA) machine learning was employed as a rapid classification method for Podolica milk recognition against the milk of other bovine species. The results of the LAP-MALDI MS analysis of milk coupled with the linear discriminant analysis (LDA) demonstrate the potential of distinguishing between Podolica and control milk samples based on MS profiles. The classification accuracy achieved in the training set is 86% while it reaches 98.4% in the test set. Bottom-up proteomics revealed approximately 220 quantified bovine proteins (identified using the Bos taurus database), with cathelicidins and annexins exhibiting higher abundance levels in control cows (intensive farming breeds). On the other hand, the metaproteomics analysis highlighted the diversity within the milk's microbial ecosystem with interesting results that may reflect the diverse environmental variables. The bottom-up proteomics data analysis using the Comprehensive Antibiotic Resistance Database (CARD) revealed beta-lactamases and tetracycline resistance proteins in both control and Podolica milk samples, with no relevant breed-specific differences observed.

Exposure to doxycycline increases risk of carrying a broad range of enteric antimicrobial resistance determinants in an elderly cohort

High rates of antibiotic prescription in residential aged care are likely to promote enteric carriage of antibiotic-resistant pathogens and increase the risk of antibiotic treatment failure. Despite their importance, relationships between antibiotic exposures and patterns of enteric resistance carriage in this population remain poorly understood. We conducted a cross-sectional metagenomic cohort analysis of stool samples from residents of five long-term aged-care facilities in South Australia. Taxonomic composition was determined, and enteric carriage of antibiotic resistance genes (ARGs) was identified and quantified against the Comprehensive Antibiotic Resistance Database. Both the detection and abundance of stool taxa and ARGs were related to antibiotic exposures up to 12 months prior. Factors associated with the abundance of ARGs of high clinical concern were identified. Stool samples were provided by 164 participants (median age: 88 years, IQR 81-93; 72% female). Sixty-one percent (n=100) of participants were prescribed antibiotics at least once in the prior 12 months (median prescriptions: 4, range: 1-52), most commonly a penicillin (n=55, 33.5%), cephalosporin (n=53, 32.3%), diaminopyrimidine (trimethoprim) (n=36, 22%), or tetracycline (doxycycline) (n=21, 12.8%). More than 1100 unique ARGs, conferring resistance to 38 antibiotic classes, were identified, including 20 ARGs of high clinical concern. Multivariate logistic regression showed doxycycline exposure to be the greatest risk factor for high ARG abundance (adjusted odds ratio [aOR]=14.8, q<0.001) and a significant contributor to inter-class selection, particularly for ARGs relating to penicillins (aOR=3.1, q=0.0004) and cephalosporins (aOR=3.4, q=0.003). High enteric ARG abundance was associated with the number of separate antibiotic exposures (aOR: 6.4, q<0.001), exposures within the prior 30 days (aOR: 4.6, q=0.008) and prior 30-100 days (aOR: 2.6, q=0.008), high duration of antibiotic exposure (aOR: 7.9, q<0.001), and exposure to 3 or more antibiotic classes (aOR: 7.4, q<0.001). Carriage of one or more ARGs of high clinical concern was identified in 99% of participants (n=162, median: 3, IQR: 2-4), involving 11 ARGs conferring resistance to aminoglycosides, four to beta-lactams, one to glycopeptides, three to fluoroquinolones, and one to oxazolidinones. Carriage of ARGs of high clinical concern was positively associated with exposure to doxycycline (aminoglycoside, fluoroquinolone, and oxazolidinone ARGs) and trimethoprim (fluoroquinolone and beta-lactam ARGs). Analysis of doxycycline impact on microbiota composition suggested that observed resistome changes arose principally through direct ARG selection, rather than through the antibiotic depletion of sensitive bacterial populations. The gut microbiome of aged care residents is a major reservoir of antibiotic resistance. As a critical antibiotic in medical practice, a comprehensive understanding of the impact of doxycycline exposure on the gut resistome is paramount for informed antibiotic use, particularly in an evolving landscape of prophylactic applications. Near-universal asymptomatic carriage of clinically critical resistance determinants is highly concerning and reinforces the urgent need for improved management of antibiotic use in long-term aged care.

Resistome Signature and Antibiotic Resistance Mechanisms in Rhizospheric Soil Bacteriomes of Mecca Region, Saudi Arabia: Insights into Impact on Human Health.

The objective of this investigation is to ascertain the distinctive profile of the rhizospheric soil resistome within the Mecca region, while also evaluating the potential risks associated with the horizontal transfer of resistome determinants to the open environment and human clinical isolates. We have made metagenomic whole-genome shotgun sequencing for rhizospheric microbiomes of two endemic plants, namely Moringa oleifera and Abutilon fruticosum. The rhizospheric resistomes of the two plants and the abundance of antibiotic resistance genes (ARGs) were identified by cross-referencing encoded proteins with the comprehensive antibiotic resistance database (CARD). The identified ARGs were then analyzed for their antimicrobial resistance (AMR) mechanisms. Predominantly within this soil are the two bacterial species Pseudomonas aeruginosa and Mycobacterium tuberculosis. These opportunistic human pathogens are implicated in respiratory infections and are correlated with heightened mortality rates. The most prevalent array of ARGs existing in this soil comprises mexA, mexC, mexE, and cpxR, associated with mechanisms of antibiotic active efflux, along with ACC(2), ACC(3), AAC(6), and APH(6), in addition to arr1, arr3, arr4, iri, rphA, and rphB, implicated in antibiotic inactivation. Furthermore, vanS, vanR, and vanJ are identified for antibiotic target alteration, while rpoB2 and RbpA are noted for antibiotic target replacement and protection, respectively. These mechanisms confer resistance against a diverse spectrum of drug classes encompassing fluoroquinolones, aminoglycosides, glycopeptides, and rifampicins. This study underscores the potential hazards posed to human health by the presence of these pathogenic bacteria within the rhizospheric soil of the Mecca region, particularly in scenarios where novel ARGs prevalent in human populations are harbored and subsequently transmitted through the food chain to human clinical isolates. Consequently, stringent adherence to good agricultural and food transportation practices is imperative, particularly with regard to edible plant parts and those utilized in folkloric medicine.

Comparative analysis of three plasmids from Plesiomonas shigelloides strain MS-17-188 and their role in antimicrobial resistance.

Plesiomonas shigelloides strain MS-17-188 was isolated from a deceased catfish from East Mississippi and showed resistance to florfenicol, tetracyclines and a sulphonamide. WGS of strain MS-17-188 revealed three plasmids (pPSMS-171881, pPSMS-171882 and pPSMS-171883). To accurately determine the impact of three plasmids found in P. shigelloides strain MS-17-188 on the dissemination of antibiotic resistance genes and to provide insights into the molecular structure of these plasmids. The genetic features of these plasmids in terms of genes associated with antimicrobial resistance (AMR), virulence, transfer, maintenance and replication were identified using bioinformatic tools. Additionally, we investigated the in vitro mobilization and stability of plasmid-mediated resistance. The Comprehensive Antibiotic Resistance Database and Virulence Factors Database were used to detect the AMR genes and virulence genes of P. shigelloides plasmids. Moreover, plasmid mobility was evaluated by a filter-mating assay using strain MS-17-188 as a donor and azide-resistant Escherichia coli J53 as a recipient strain. A stability experiment was conducted to explore the persistence of plasmid-mediated antibiotic resistance in strain MS-17-188 in the absence and presence of selection. pPSMS-171881 harboured multidrug efflux complex (adeF) and two genes responsible for arsenic resistance (arsB and arsC). pPSMS-171882 had a region of 7085 bp encoding type IV secretion system proteins. pPSMS-171883 carried the tetracycline resistance genes tet(A) and tet(R), and a phenicol resistance gene (floR), which were flanked by two transposable elements and mobilization proteins, suggesting that there is a conjugative mechanism by which this plasmid can be mobilized. Results from the stability experiment indicated that pPSMS-171883 is lost over time in the absence of selective pressure. Moreover, pPSMS-171883 is more stable in P. shigelloides at growth temperatures of 30°C and 37°C compared with 40°C and 43°C. After intraperitoneal injection in catfish, P. shigelloides strain MS-17-188 resulted in no mortalities. This is the first study to report plasmid-mediated AMR in Plesiomonas isolated from cultured fish, which needs continued monitoring. This study will provide an understanding of the genetic mechanisms of AMR and virulence of P. shigelloides.

Metagenomics Analysis Reveals the Composition and Functional Differences of Fecal Microbiota in Wild, Farm, and Released Chinese Three-Keeled Pond Turtles (Mauremys reevesii).

The intestine of living organisms harbors different microbiota associated with the biological functioning and health of the host and influences the process of ecological adaptation. Here, we studied the intestinal microbiota's composition and functional differences using 16S rRNA and metagenomic analysis in the wild, farm, and released Chinese three-keeled pond turtle (Mauremys reevesii). At the phylum level, Bacteroidota dominated, followed by Firmicutes, Fusobacteriota, and Actinobacteriota in the wild group, but Chloroflexi was more abundant in the farm and released groups. Moreover, Chryseobacterium, Acinetobacter, Comamonas, Sphingobacterium, and Rhodobacter were abundant in the released and farm cohorts, respectively. Cetobacterium, Paraclostridium, Lysobacter, and Leucobacter showed an abundance in the wild group. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed that the relative abundance of most pathways was significantly higher in the wild turtles (carbohydrate metabolism, lipid metabolism, metabolism of cofactors, and vitamins). The comprehensive antibiotic resistance database (CARD) showed that the antibiotic resistance gene (ARG) subtype macB was the most abundant in the farm turtle group, while tetA was higher in the wild turtles, and srpYmcr was higher in the released group. Our findings shed light on the association between the intestinal microbiota of M. reevesii and its habitats and could be useful for tracking habitats to protect and conserve this endangered species.

Investigating gut microbiota-blood and urine metabolite correlations in early sepsis-induced acute kidney injury: insights from targeted KEGG analyses.

The interplay between gut microbiota and metabolites in the early stages of sepsis-induced acute kidney injury (SA-AKI) is not yet clearly understood. This study explores the characteristics and interactions of gut microbiota, and blood and urinary metabolites in patients with SA-AKI. Utilizing a prospective observational approach, we conducted comparative analyses of gut microbiota and metabolites via metabolomics and metagenomics in individuals diagnosed with SA-AKI compared to those without AKI (NCT06197828). Pearson correlations were used to identify associations between microbiota, metabolites, and clinical indicators. The Comprehensive Antibiotic Resistance Database was employed to detect antibiotic resistance genes (ARGs), while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways informed on metabolic processes and microbial resistance patterns. Our study included analysis of four patients with SA-AKI and five without AKI. Significant disparities in bacterial composition were observed, illustrated by diversity indices (Shannon index: 2.0 ± 0.4 vs. 1.4 ± 0.6, P = 0.230; Simpson index: 0.8 ± 0.1 vs. 0.6 ± 0.2, P = 0.494) between the SA-AKI group and the non-AKI group. N6, N6, N6-Trimethyl-L-lysine was detected in both blood and urine metabolites, and also showed significant correlations with specific gut microbiota (Campylobacter hominis and Bacteroides caccae, R > 0, P < 0.05). Both blood and urine metabolites were enriched in the lysine degradation pathway. We also identified the citrate cycle (TCA cycle) as a KEGG pathway enriched in sets of differentially expressed ARGs in the gut microbiota, which exhibits an association with lysine degradation. Significant differences in gut microbiota and metabolites were observed between the SA-AKI and non-AKI groups, uncovering potential biomarkers and metabolic changes linked to SA-AKI. The lysine degradation pathway may serve as a crucial link connecting gut microbiota and metabolites.

Alterations of mouse gut microbiome in alveolar echinococcosis

Alveolar echinococcosis (AE) may affect the composition of the host's gut microbiota, potentially disrupting the balance between the gut microbiota and metabolites. Metagenomics and untargeted metabolomics were employed to characterize changes in the gut microbiota and metabolites in mouse models infected with E. multilocularis. Pearson correlation coefficients were calculated to compare the distribution of microbiota and metabolites, revealing synergistic or mutually exclusive relationships. Functional outputs of the gut microbiota were explored using the CAZy database and six enzymes involved in carbohydrate metabolism were identified with statistically significant differential expression between infected and control groups. The resistome was characterized by identifying antibiotic resistance genes annotated in the Comprehensive Antibiotic Resistance Database from the metagenomes of the groups. Firmicutes are the main carrier of ARGs in the host gut with tetQ being most prevalent. Antibiotic efflux, inactivation and target modification were the principal mechanisms of resistance. Comparison and analysis of two sets of antibiotic metabolic pathways allowed the identification of enzyme reactions unique to infected mice. KEGG pathway overview shows phenazine biosynthesis involving phzG to be one of them. In conclusion, infection with AE in mice leads to an overall disruption of gut microbiota and metabolites with the involvement of enzymes related to carbohydrate metabolism. Furthermore, antibiotic-resistance genes may play a role in disease progression, offering potential insights into the relationship between antibiotic use in AE and treatment outcomes.

Phenotypic and genotypic analysis of antimicrobial resistance and population structure of gastroenteritis-related Aeromonas isolates

BackgroundThe population structure and the correlation between antimicrobial resistance (AMR) phenotypes and genotypes in Aeromonas species isolated from patients with gastroenteritis are not well understood. The aims of the study were to: (1) investigate the antimicrobial susceptibility profiles of Aeromonas species isolated from patients with gastroenteritis; (2) explore the relationship between AMR genes and resistance phenotypes; and (3) describe the population structure of these isolates and provide evidence of transmission events among them.MethodsThis microbiological survey was performed at the Microbiology Laboratory of the Emek Medical Center in Afula, Israel. Cultivation of Aeromonas was attempted from stool samples that tested positive by PCR. Antimicrobial susceptibility testing (AST) was performed using the Sensititre GN3F microdilution panel. Whole genome sequencing (WGS) was done using the Illumina NextSeq500/550 system. Phylogenetic studies involved multi-locus sequence typing (MLST) and core genome (cg) MLST. Resistance mechanisms were identified using the Comprehensive Antibiotic Resistance Database and compared with the AST results.ResultsThe study included 67 patient-unique isolates. The species that were identified included A. caviae (n = 58), A. dhakensis (n = 3), A. media (n = 2), A. veronii (n = 2) and A. hydrophila (n = 2). Isolates were almost uniformly susceptible to amikacin, gentamicin, aztreonam, cefepime, ceftazidime, ciprofloxacin and meropenem. All isolates with the exception of 1–2 isolates were resistant to ampicillin, cefazolin and ampicillin-sulbactam which was compatible with the presence of the blaOXA genes. Variable resistance rates were observed to cefuroxime, cefoxitin, ceftriaxone, piperacillin-tazobactam that were not correlated with the presence of other β-lactamase genes. Resistance to tetracycline and trimethoprim-sulfamethoxazole correlated with the presence of tetA and sul1, respectively. The population structure of A. caviae was highly diverse with the minority of the isolates (16/57) clustering into six defined sequence types. A cgMLST-based distance of four genes was found in one pair of isolates, suggesting common source transmission.ConclusionsA. caviae is the dominant species related to gastroenteritis and is characterized by a diverse population structure, with almost no evidence for common-source transmission. Resistance rates to most antimicrobial agents were low and partially matched with the presence of resistance genes.

Metagenomic and Antibiotic Resistance Analysis of the Gut Microbiota in Larus relictus and Anatidae Species Inhabiting the Honghaizi Wetland of Ordos, Inner Mongolia, from 2021 to 2023.

Gut microbes thrive by utilising host energy and, in return, provide valuable benefits, akin to a symbiotic relationship. Here, metagenomic sequencing was performed to characterise and compare the community composition, diversity and antibiotic resistance of the gut microbiota of Relict gull (Larus relictus) and Anatidae species. Alpha diversity analysis revealed that the intestinal microbial richness of L. relictus was significantly lower than that of Anatidae, with distinct differences observed in microbial composition. Notably, the intestines of L. relictus harboured more pathogenic bacteria such as clostridium, which may contribute to the decline in their population and endangered status. A total of 117 strains of Escherichia coli were isolated, with 90.60% exhibiting full susceptibility to 21 antibiotics, while 25.3% exhibited significant biofilm formation. Comprehensive Antibiotic Resistance Database data indicated that glycopeptide resistance genes were the most prevalent type carried by migratory birds, alongside quinolone, tetracycline and lincosamide resistance genes. The abundance of resistance genes carried by migratory birds decreased over time. This metagenomic analysis provides valuable insights into the intestinal microbial composition of these wild bird species, offering important guidance for their conservation efforts, particularly for L. relictus, and contributing to our understanding of pathogen spread and antibiotic-resistant bacteria.

Influence of helicobacter pylori on composition and function of gastric microbiota in patients with chronic non-atrophic gastritis

ObjectiveHelicobacter pylori (H. pylori) plays a major role in causing and advancing gastrointestinal illnesses. Our aim is to analyze the unique makeup and functional changes in the gastric microbiota of patients with chronic non-atrophic gastritis (CNAG), regardless of the presence of H. pylori, and to determine the potential signaling pathways. MethodsWe performed metagenomic sequencing on gastric mucosa samples collected from 17 individuals with non-atrophic gastritis, comprising 6 cases were infected with H. pylori (H. pylori-infected case group) and 11 cases without (control group). The species composition was evaluated with DIAMOND software, and functional enrichment was assessed utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We analyzed antibiotic resistance patterns using the Comprehensive Antibiotic Resistance Database as a reference (CARD). ResultsThe presence of H. pylori colonization in CNAG patients was associated with increased diversity in the gastric microbiota. The Phylum Firmicutes was found to be less prevalent, while the Phylum Proteobacteria showed an increase. Functionally, pathways associated with metabolic pathways, including vitamins, auxiliaries, amino acid residue, carbon hydrate, and metabolic energy pathways, were enriched in CNAG patients with H. pylori infection. Additionally, antibiotic resistance genes correlated with antibiotic efflux pump were enriched. ConclusionsFrom a holistic genomic perspective, our findings offer fresh perspectives into the gastric microbiome among CNAG patients carrying H. pylori, which is valuable for future research on CNAG.

Metatranscriptome and Resistome of the Endodontic Microbiome

IntroductionIn this study, we used metatranscriptomics for the first time to investigate microbial composition, functional signatures, and antimicrobial resistance gene expression in endodontic infections. MethodsRoot canal samples were collected from ten teeth, including five primary and five persistent/secondary endodontic infections. RNA from endodontic samples was extracted, and RNA sequencing was performed on a NovaSeq6000 system (Illumina). Taxonomic analysis was performed using the Kraken2 bacterial database. Then, sequences with a taxonomic classification were annotated against the Universal Protein Knowledgebase for functional annotation and the Comprehensive Antibiotic Resistance Database for AR-like gene identification. ResultsProteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria represented the dominant phyla, whereas Fusobacteria, Spirochetes, and Synergistetes were among the nondominant phyla. The top ten species were mainly represented by obligate (or quasiobligate) anaerobes, including Gram-negative (eg, Capnocytophaga sp. oral taxon 323, Fusobacterium nucleatum, Prevotella intermedia, Prevotella oris, Tannerella forsythia, and Tannerella sp. oral taxon HOT−286) and Gram-positive species (eg, Olsenella uli and Parvimonas micra). Transcripts encoding moonlighting proteins (eg, glycolytic proteins, translational elongation factors, chaperonin, and heat shock proteins) were highly expressed, potentially affecting bacterial adhesion, biofilm formation, host defense evasion, and inflammation induction. Endodontic bacteria expressed genes conferring resistance to antibiotic classes commonly used in dentistry, with a high prevalence and expression of tetracycline and lincosamide resistance genes. Antibiotic efflux and antibiotic target alteration/protection were the main resistance mechanisms. ConclusionsMetatranscriptomics revealed the activity of potential endodontic pathogens, which expressed putative virulence factors and a wide diversity of genes potentially involved in AR.

Whole Genome Analysis of Tibetan Kefir-Derived Lactiplantibacillus Plantarum 12-3 Elucidates Its Genomic Architecture, Antimicrobial and Drug Resistance, Potential Probiotic Functionality and Safety.

Lactiplantibacillus plantarum 12-3 holds great promise as a probiotic bacterial strain, yet its full potential remains untapped. This study aimed to better understand this potential therapeutic strain by exploring its genomic landscape, genetic diversity, CRISPR-Cas mechanism, genotype, and mechanistic perspectives for probiotic functionality and safety applications. L. plantarum 12-3 was isolated from Tibetan kefir grains and, subsequently, Illumina and Single Molecule Real-Time (SMRT) technologies were used to extract and sequence genomic DNA from this organism. After performing pan-genomic and phylogenetic analysis, Average Nucleotide Identity (ANI) was used to confirm the taxonomic identity of the strain. Antibiotic resistance gene analysis was conducted using the Comprehensive Antibiotic Resistance Database (CARD). Antimicrobial susceptibility testing, and virulence gene identification were also included in our genomic analysis to evaluate food safety. Prophage, genomic islands, insertion sequences, and CRISPR-Cas sequence analyses were also carried out to gain insight into genetic components and defensive mechanisms within the bacterial genome. The 3.4 Mb genome of L. plantarum 12-3, was assembled with 99.1% completeness and low contamination. A total of 3234 genes with normal length and intergenic spacing were found using gene prediction tools. Pan-genomic studies demonstrated gene diversity and provided functional annotation, whereas phylogenetic analysis verified taxonomic identity. Our food safety study revealed a profile of antibiotic resistance that is favorable for use as a probiotic. Analysis of insertional sequences, genomic islands, and prophage within the genome provided information regarding genetic components and their possible effects on evolution. Pivotal genetic elements uncovered in this study play a crucial role in bacterial defense mechanisms and offer intriguing prospects for future genome engineering efforts. Moreover, our findings suggest further in vitro and in vivo studies are warranted to validate the functional attributes and probiotic potential of L. plantarum 12-3. Expanding the scope of the research to encompass a broader range of L. plantarum 12-3 strains and comparative analyses with other probiotic species would enhance our understanding of this organism's genetic diversity and functional properties.

A sophisticated virulence repertoire and colistin resistance of Citrobacter freundii ST150 from a patient with sepsis admitted to ICU in a tertiary care hospital in Uganda, East Africa: Insight from genomic and molecular docking analyses

Sepsis and multidrug resistance comprise a complex of factors attributable to mortality among intensive care unit (ICU) patients globally. Pathogens implicated in sepsis are diverse, and their virulence and drug resistance remain elusive. From a tertiary care hospital ICU in Uganda, we isolated a Citrobacter freundii strain RSM030 from a patient with sepsis and phenotypically tested it against a panel of 16 antibiotics including imipenem levofloxacin, cotrimoxazole and colistin, among others. We sequenced the organism's genome and integrated multilocus sequencing (MLST), PathogenFinder with Virulence Factor analyzer (VFanalyzer) to establish its pathogenic relevance. Thereafter, we combined antiSMASH and PRISM genome mining with molecular docking to predict biosynthetic gene clusters (BGCs), pathways, toxin structures and their potential targets in-silico. Finally, we coupled ResFinder with comprehensive antibiotic resistance database (CARD) to scrutinize the genomic antimicrobial resistance profile of the isolate. From PathogenFinder and MLST, this organism was confirmed to be a human pathogen (p = 0.843), sequence type (ST)150, whose virulence is determined by chromosomal type III secretion system (T3SS) (the injectosome) and plasmid-encoded type IV secretion system (T4SS), the enterobactin biosynthetic gene cluster and biofilm formation through the pgaABCD operon. Pathway and molecular docking analyses revealed that the shikimate pathway can generate a toxin targeting multiple host proteins including spectrin, detector of cytokinesis protein 2 (Dock2) and plasmalemma vesicle-associated protein (PLVAP), potentially distorting the host cell integrity. From phenotypic antibiotic testing, we found indeterminate results for amoxicillin/clavulanate and levofloxacin, with resistance to cotrimoxazole and colistin. Detailed genome analysis revealed chromosomal beta lactam resistance genes, i.e. blaCMY-79, blaCMY-116 and blaTEM-1B, along with multiple mutations of the lipopolysaccharide modifying operon genes PmrA/PmrB, pmrD, mgrA/mgrB and PhoP/PhoQ, conferring colistin resistance. From these findings, we infer that Citrobacter freundii strain RSM030 is implicated in sepsis and resistance to standard antibiotics, including colistin, the last resort.

Using nanopore sequencing to identify bacterial infection in joint replacements: a preliminary study.

This project investigates if third-generation genomic sequencing can be used to identify the species of bacteria causing prosthetic joint infections (PJIs) at the time of revision surgery. Samples of prosthetic fluid were taken during revision surgery from patients with known PJIs. Samples from revision surgeries from non-infected patients acted as negative controls. Genomic sequencing was performed using the MinION device and the rapid sequencing kit from Oxford Nanopore Technologies. Bioinformatic analysis pipelines to identify bacteria included Basic Local Alignment Search Tool, Kraken2 and MinION Detection Software, and the results were compared with standard of care microbiological cultures. Furthermore, there was an attempt to predict antibiotic resistance using computational tools including ResFinder, AMRFinderPlus and Comprehensive Antibiotic Resistance Database. Bacteria identified using microbiological cultures were successfully identified using bioinformatic analysis pipelines. Nanopore sequencing and genomic classification could be completed in the time it takes to perform joint revision surgery (2-3h). Genomic sequencing in this study was not able to predict antibiotic resistance in this time frame, this is thought to be due to a short-read length and low read depth. It can be concluded that genomic sequencing can be useful to identify bacterial species in infected joint replacements. However, further work is required to investigate if it can be used to predict antibiotic resistance within clinically relevant timeframes.

Emergence and clonal expansion of Aeromonas hydrophila ST1172 that simultaneously produces MOX-13 and OXA-724

BackgroundAeromonas hydrophila infections can cause gastrointestinal symptoms such as diarrhea; however, deep infections are rarely reported. Outbreaks of A. hydrophila are reported more frequently in fish, poultry, and snakes than in humans. This study aimed to track clonal relatedness of deep infections caused by A. hydrophila using whole genome sequencing (WGS).MethodsWe collected three isolates of A. hydrophila in July 19 to August 29, 2019, from patients that underwent spine surgery. Accurate species identification was performed using whole-genome average nucleotide identity (ANI). Antimicrobial susceptibility testing was performed using a VITEK 2 automated AST-N334 Gram-negative susceptibility card system. Antimicrobial resistance and virulence genes were identified using the Comprehensive Antibiotic Resistance Database and Virulence Factor Database VFanalyzer.ResultsAll three isolates were identified as A. hydrophila based on ANI and multilocus sequence typing analysis revealed that A. hydrophila belonged to a novel sequence type (ST1172). All three isolates were susceptible to amikacin and levofloxacin; however, they were resistant to piperacillin/tazobactam, ceftriaxone, cefuroxime, cefoxitin, and imipenem. Isolate 19W05620 (patient 3) showed increased ceftazidime resistance (minimum inhibitory concentration ≥ 64 µg/mL). All three isolates possessed the same chromosomally encoded β-lactamases, including blaOXA-724 (β-lactamase), imiH (metallo-β-lactamase), and blaMOX-13 (AmpC) in plasmids.ConclusionsOur study validated the transmission of a novel carbapenem-resistant A. hydrophila sequence type (ST1172) in patients that underwent spine surgery. Control measures should be developed to prevent dissemination of A. hydrophila in the hospital setting.

IS6 family insertion sequences promote optrA dissemination between plasmids varying in transfer abilities.

Plasmids are the primary vectors for intercellular transfer of the oxazolidinone and phenicol cross-resistance gene optrA, while insertion sequences (ISs) are mobile genetic elements that can mobilize plasmid-borne optrA intracellularly. However, little is known about how the IS-mediated intracellular mobility facilitates the dissemination of the optrA gene between plasmid categories that vary in transfer abilities, including non-mobilizable, mobilizable, and conjugative plasmids. Here, we performed a holistic genomic study of 52 optrA-carrying plasmids obtained from searches guided by the Comprehensive Antibiotic Resistance Database. Among the 132 ISs identified within 10 kbp from the optrA gene in the plasmids, IS6 family genes were the most prevalent (86/132). Homologous gene arrays containing IS6 family genes were shared between different plasmids, especially between mobilizable and conjugative plasmids. All these indicated the central role of IS6 family genes in disseminating plasmid-borne optrA. Thirty-three of the 52 plasmids were harbored by Enterococcus faecalis found mainly in humans and animals. By Nanopore sequencing and inverse PCR, the potential of the enterococcal optrA to be transmitted from a mobilizable plasmid to a conjugative plasmid mediated by IS6 family genes was further confirmed in Enterococcus faecalis strains recovered from the effluents of anaerobic digestion systems for treating chicken manure. Our findings highlight the increased intercellular transfer abilities and dissemination risk of plasmid-borne optrA gene caused by IS-mediated intracellular mobility, and underscore the importance of routinely monitoring the dynamic genetic contexts of clinically important antibiotic resistance genes to effectively control this critical public health threat. KEY POINTS: • IS6 was prevalent in optrA-plasmids varying in intercellular transfer abilities. • Enterococcal optrA-plasmids were widespread among human, animal, and the environment. • IS6 elevated the dissemination risk of enterococcal optrA-plasmids.

Safety assessment of five candidate probiotic lactobacilli using comparative genome analysis.

Micro-organisms belonging to the Lactobacillus genus complex are often used for oral consumption and are generally considered safe but can exhibit pathogenicity in rare and specific cases. Therefore, screening and understanding genetic factors that may contribute to pathogenicity can yield valuable insights regarding probiotic safety. Limosilactobacillus mucosae LM1, Lactiplantibacillus plantarum SK151, Lactiplantibacillus plantarum BS25, Limosilactobacillus fermentum SK152 and Lactobacillus johnsonii PF01 are current probiotics of interest; however, their safety profiles have not been explored. The genome sequences of LM1, SK151, SK152 and PF01 were downloaded from the NCBI GenBank, while that of L. plantarum BS25 was newly sequenced. These genomes were then annotated using the Rapid Annotation using Subsystem Technology tool kit pipeline. Subsequently, a command line blast was performed against the Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD) to identify potential virulence factors and antibiotic resistance (AR) genes. Furthermore, ResFinder was used to detect acquired AR genes. The query against the VFDB identified genes that have a role in bacterial survivability, platelet aggregation, surface adhesion, biofilm formation and immunoregulation; and no acquired AR genes were detected using CARD and ResFinder. The study shows that the query strains exhibit genes identical to those present in pathogenic bacteria with the genes matched primarily having roles related to survival and surface adherence. Our results contribute to the overall strategies that can be employed in pre-clinical safety assessments of potential probiotics. Gene mining using whole-genome data, coupled with experimental validation, can be implemented in future probiotic safety assessment strategies.