RESUMEN
Pseudomonas aeruginosa Y010, isolated from the taro rhizosphere, exhibits great antagonistic abilities against Dickeya strains that cause soft-rot and blackleg diseases of plants by producing potent antimicrobial agents. The complete genome of Y010 was sequenced and annotated, which is 6,415,628 bp in length with 66.39% GC content.
RESUMEN
The NG-Test CARBA 5 and Carbapenem-resistant K.N.I.V.O. Detection K-Set are lateral flow assays (LFAs) that rapidly detect five carbapenemases (KPC, NDM, IMP, VIM and OXA-48-like). We evaluated the effect of inoculum size on the performance of these two assays using 27 Enterobacterales isolates. Whole-genome sequencing (WGS) was used as the reference method. Using the NG-Test CARBA 5, eight Serratia spp. and six M. morganii isolates showed false-positive NDM results with a high inoculum. Using the Carbapenem-resistant K.N.I.V.O. Detection K-Set, eight M. morganii, four Serratia spp. and one K. pneumoniae isolates showed false-positive NDM and/or OXA-48-like bands at large inoculum sizes, while the other two M. morganii isolates demonstrated false-positive NDM and OXA-48-like results at all inoculum sizes. The false-positive bands varied in intensity. WGS confirmed that no carbapenemase gene was present. No protein sequence with a ≥50% identity to NDM or OXA-48-like enzymes was found. This study emphasizes the importance of assessing inoculum size in the diagnostic evaluation of LFAs.
RESUMEN
Lactococcus garvieae is a fish pathogen that can cause diseases in humans and cows. Two genetically related species, Lactococcus formosensis and Lactococcus petauri, may be misidentified as L. garvieae. It is unclear if these species differ in host specificity and virulence genes. This study analyzed the genomes of 120 L. petauri, 53 L. formosensis, and 39 L. garvieae isolates from various sources. The genetic diversity and virulence gene content of these isolates were compared. The results showed that 77 isolates previously reported as L. garvieae were actually L. formosensis or L. petauri. The distribution of the three species varied across different collection sources, with L. petauri being predominant in human infections, human fecal sources, and rainbow trout, while L. formosensis was more common in bovine isolates. The genetic diversity of isolates within each species was high and similar. Using a genomic clustering method, L. petauri, L. formosensis, and L. garvieae were divided into 45, 22, and 13 clusters, respectively. Most rainbow trout and human isolates of L. petauri belonged to different clusters, while L. formosensis isolates from bovine and human sources were also segregated into separate clusters. In L. garvieae, most human isolates were grouped into three clusters that also included isolates from food or other sources. Non-metric multidimensional scaling ordination revealed the differential association of 15 virulence genes, including 14 adherence genes and a bile salt hydrolase gene, with bacterial species and certain collection sources. In conclusion, this work provides evidence of host specificity among the three species. IMPORTANCE: Lactococcus formosensis and Lactococcus petauri are two newly discovered bacteria, which are closely related to Lactococcus garvieae, a pathogen that affects farmed rainbow trout, as well as causes cow mastitis and human infections. It is unclear whether the three bacteria differ in their host preference and the presence of genes that contribute to the development of disease. This study shows that L. formosensis and L. petauri were commonly misidentified as L. garvieae. The three bacteria showed different distribution patterns across various sources. L. petauri was predominantly found in human infections and rainbow trout, while L. formosensis was more commonly detected in cow mastitis. Fifteen genes displayed a differential distribution among the three bacteria from certain sources, indicating a genetic basis for the observed host preference. This work indicates the importance of differentiating the three bacteria in diagnostic laboratories for surveillance and outbreak investigation purposes.
Asunto(s)
Variación Genética , Genoma Bacteriano , Especificidad del Huésped , Lactococcus , Animales , Lactococcus/genética , Lactococcus/clasificación , Lactococcus/aislamiento & purificación , Humanos , Bovinos , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/veterinaria , Factores de Virulencia/genética , Filogenia , Oncorhynchus mykiss/microbiología , Genómica , Virulencia/genética , Heces/microbiologíaRESUMEN
Metagenomic binning is an essential technique for genome-resolved characterization of uncultured microorganisms in various ecosystems but hampered by the low efficiency of binning tools in adequately recovering metagenome-assembled genomes (MAGs). Here, we introduce BASALT (Binning Across a Series of Assemblies Toolkit) for binning and refinement of short- and long-read sequencing data. BASALT employs multiple binners with multiple thresholds to produce initial bins, then utilizes neural networks to identify core sequences to remove redundant bins and refine non-redundant bins. Using the same assemblies generated from Critical Assessment of Metagenome Interpretation (CAMI) datasets, BASALT produces up to twice as many MAGs as VAMB, DASTool, or metaWRAP. Processing assemblies from a lake sediment dataset, BASALT produces ~30% more MAGs than metaWRAP, including 21 unique class-level prokaryotic lineages. Functional annotations reveal that BASALT can retrieve 47.6% more non-redundant opening-reading frames than metaWRAP. These results highlight the robust handling of metagenomic sequencing data of BASALT.
Asunto(s)
Ecosistema , Metagenoma , Silicatos , Metagenoma/genética , Metagenómica/métodosRESUMEN
Zinc is an important transition metal that is essential for numerous physiological processes while excessive zinc is cytotoxic. Pseudomonas aeruginosa is a ubiquitous opportunistic human pathogen equipped with an exquisite zinc homeostatic system, and the two-component system CzcS/CzcR plays a key role in zinc detoxification. Although an increasing number of studies have shown the versatility of CzcS/CzcR, its physiological functions are still not fully understood. In this study, transcriptome analysis was performed, which revealed that CzcS/CzcR is silenced in the absence of the zinc signal but modulates global gene expression when the pathogen encounters zinc excess. CzcR was demonstrated to positively regulate the copper tolerance gene ptrA and negatively regulate the pyochelin biosynthesis regulatory gene pchR through direct binding to their promoters. Remarkably, the upregulation of ptrA and downregulation of pchR were shown to rescue the impaired capacity of copper tolerance and prevent pyochelin overproduction, respectively, caused by zinc excess. This study not only advances our understanding of the regulatory spectrum of CzcS/CzcR but also provides new insights into stress adaptation mediated by two-component systems in bacteria to balance the cellular processes that are disturbed by their signals. IMPORTANCE: CzcS/CzcR is a two-component system that has been found to modulate zinc homeostasis, quorum sensing, and antibiotic resistance in Pseudomonas aeruginosa. To fully understand the physiological functions of CzcS/CzcR, we performed a comparative transcriptome analysis in this study and discovered that CzcS/CzcR controls global gene expression when it is activated during zinc excess. In particular, we demonstrated that CzcS/CzcR is critical for maintaining copper tolerance and iron homeostasis, which are disrupted during zinc excess, by inducing the expression of the copper tolerance gene ptrA and repressing the pyochelin biosynthesis genes through pchR. This study revealed the global regulatory functions of CzcS/CzcR and described a new and intricate adaptive mechanism in response to zinc excess in P. aeruginosa. The findings of this study have important implications for novel anti-infective interventions by incorporating metal-based drugs.
Asunto(s)
Cobre , Fenoles , Infecciones por Pseudomonas , Tiazoles , Humanos , Cobre/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Zinc/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
Pseudomonas aeruginosa has abundant signaling systems that exquisitely control its antibiotic resistance in response to different environmental cues. Understanding the regulation of antibiotic resistance will provide important implications for precise antimicrobial interventions. However, efficient genetic tools for functional gene characterizations are sometimes not available, particularly, in clinically isolated strains. Here, we established a type I-F CRISPRi (CSYi) system for programmable gene silencing. By incorporating anti-CRISPR proteins, this system was even applicable to bacterial hosts encoding a native type I-F CRISPR-Cas system. With the newly developed gene-silencing system, we revealed that the response regulator CzcR from the zinc (Zn2+)-responsive two-component system CzcS/CzcR is a repressor of efflux pumps MexAB-OprM and MexGHI-OpmD, which inhibits the expression of both operons by directly interacting with their promoters. Repression of MexAB-OprM consequently increases the susceptibility of P. aeruginosa to multiple antibiotics such as levofloxacin and amikacin. Together, this study provided a simple approach to study gene functions, which enabled us to unveil the novel role of CzcR in modulating efflux pump genes and multidrug resistance in P. aeruginosa. IMPORTANCE P. aeruginosa is a ubiquitous opportunistic pathogen frequently causing chronic infections. In addition to being an important model organism for antibiotic-resistant research, this species is also important for understanding and exploiting CRISPR-Cas systems. In this study, we established a gene-silencing system based on the most abundant type I-F CRISPR-Cas system in this species, which can be readily employed to achieve targeted gene repression in multiple bacterial species. Using this gene-silencing system, the physiological role of Zn2+ and its responsive regulator CzcR in modulating multidrug resistance was unveiled with great convenience. This study not only displayed a new framework to expand the abundant CRISPR-Cas and anti-CRISPR systems for functional gene characterizations but also provided new insights into the regulation of multidrug resistance in P. aeruginosa and important clues for precise anti-pseudomonal therapies.
RESUMEN
Introduction: Polymyxin-resistant Enterobacterales poses a significant threat to public health globally, but its prevalence and genomic diversity within a sole hospital is less well known. In this study, the prevalence of polymyxin-resistant Enterobacterales in a Chinese teaching hospital was investigated with deciphering of their genetic determinants of drug resistance. Methods: Polymyxin-resistant Enterobacterales isolates identified by matrix-assisted laser desorption were collected in Ruijin Hospital from May to December in 2021. Both the VITEK 2 Compact and broth dilution methods were used to determine polymyxin B (PMB) susceptibility. Polymyxin-resistant isolates were further characterized by molecular typing using PCR, multi-locus sequence typing, and sequencing of the whole genome. Results: Of the 1,216 isolates collected, 32 (2.6%) across 12 wards were polymyxin-resistant (minimum inhibitory concentration (MIC) range, PMB 4-256 mg/ml, and colistin 4 ≥ 16 mg/ ml). A total of 28 (87.5%) of the polymyxin-resistant isolates had reduced susceptibility to imipenem and meropenem (MIC ≥ 16 mg/ml). Of the 32 patients, 15 patients received PMB treatment and 20 survived before discharge. The phylogenetic tree of these isolates showed they belonged to different clones and had multiple origins. The polymyxin-resistant Klebsiella pneumoniae isolates belonged to ST-11 (85.72%), ST-15 (10.71%), and ST-65 (3.57%), and the polymyxin-resistant Escherichia coli belonged to four different sequence types, namely, ST-69 (25.00%), ST-38 (25.00%), ST-648 (25.00%), and ST-1193 (25.00%). In addition, six mgrB specific mutations (snp_ALT c.323T>C and amino acid change p.Val8Ala) were identified in 15.6% (5/32) of the isolates. mcr-1, a plasmid-mediated polymyxin-resistant gene, was found in three isolates, and non-synonymous mutations including T157P, A246T, G53V, and I44L were also observed. Discussion: In our study, a low prevalence of polymyxin-resistant Enterobacterales was observed, but these isolates were also identified as multidrug resistant. Therefore, efficient infection control measures should be implemented to prevent the further spread of resistance to last-line polymyxin therapy.
Asunto(s)
Proteínas de Escherichia coli , Polimixinas , Humanos , Polimixinas/farmacología , Antibacterianos/farmacología , Tipificación de Secuencias Multilocus , Prevalencia , Filogenia , Pueblos del Este de Asia , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Hospitales de Enseñanza , Klebsiella pneumoniae , Pruebas de Sensibilidad Microbiana , Proteínas de Escherichia coli/genéticaRESUMEN
Typhoid fever is a notable disease in Hong Kong. We noticed two local cases of typhoid fever caused by Salmonella Typhi within a two-week period in late 2022, which had no apparent epidemiological linkage except for residing in the same region of Hong Kong. A phylogenetic study of Salmonella Typhi isolates from Hong Kong Island from 2020 to 2022 was performed, including a whole-genome analysis, the typing of plasmids, and the analysis of antibiotic-resistance genes (ARGs), to identify the dominant circulating strain and the spread of ARGs. A total of seven isolates, from six local cases and an imported case, were identified from positive blood cultures in two hospitals in Hong Kong. Five antibiotic-sensitive strains of genotype 3.2.2 were found, which clustered with another 30 strains originating from Southeast Asia. Whole-genome sequencing revealed clonal transmission between the two index cases. The remaining two local cases belong to genotype 2.3.4 and genotype 4.3.1.1.P1 (also known as the H58 lineage). The genotype 4.3.1.1.P1 strain has an extensively drug-resistant (XDR) phenotype (co-resistance to ampicillin, chloramphenicol, ceftriaxone, ciprofloxacin, and co-trimoxazole). Although the majority of local strains belong to the non-H58 genotype 3.2.2 with a low degree of antibiotic resistance, the introduction of XDR strains with the global dissemination of the H58 lineage remains a concern.
RESUMEN
Bacteremia caused by extended-spectrum ß-lactamases-producing Enterobacterales has increased rapidly and is mainly attributed to CTX-M enzymes. This study aimed to evaluate the NG-Test® CTX-M MULTI lateral flow assay (CTX-M LFA) for rapid detection of CTX-M producers in blood cultures (BCs) positive for Gram-negative bacilli in spiked and clinical BCs. Retrospective testing was performed on BC bottles spiked with a collection of well-characterized Enterobacterales isolates producing CTX-M (n = 15) and CTX-M-like (n = 27) ß-lactamases. Prospective testing of clinical, non-duplicate BCs (n = 350) was performed in two hospital microbiology laboratories from April 2021 to March 2022 following detection of Gram-negative bacilli by microscopic examination. Results were compared against molecular testing as the reference. In the spiked BCs, the CTX-M LFA correctly detected all CTX-M producers including 5 isolates with hybrid CTX-M variants. However, false-positive results were observed for several CTX-M-like ß-lactamases, including OXY-1-3, OXY-2-8, OXY-5-3, FONA-8, -9, -10, 11, 13 and SFO-1. In clinical BCs, the CTX-M LFA showed 100% (95% CI, 96.0-100%) sensitivity and 99.6% (97.9-100%) specificity. In conclusion, this study showed that rapid detection of CTX-M producers in BC broths can be reliably achieved using the CTX-M LFA, thus providing an opportunity for early optimization of antibiotics.
RESUMEN
Natural products (NPs) especially the secondary metabolites originated from microbes exhibit great importance in biomedical, industrial and agricultural applications. However, mining biosynthetic gene clusters (BGCs) to produce novel NPs has been hindered owing that a large population of environmental microbes are unculturable. In the past decade, strategies to explore BGCs directly from (meta)genomes have been established along with the fast development of high-throughput sequencing technologies and the powerful bioinformatics data-processing tools, which greatly expedited the exploitations of novel BGCs from unculturable microbes including the extremophilic microbes. In this review, we firstly summarized the popular bioinformatics tools and databases available to mine novel BGCs from (meta)genomes based on either pure cultures or pristine environmental samples. Noticeably, approaches rooted from machine learning and deep learning with focuses on the prediction of ribosomally synthesized and post-translationally modified peptides (RiPPs) were dramatically increased in recent years. Moreover, synthetic biology techniques to express the novel BGCs in culturable native microbes or heterologous hosts were introduced. This working pipeline including the discovery and biosynthesis of novel NPs will greatly advance the exploitations of the abundant but unexplored microbial BGCs.
Asunto(s)
Biología Computacional , Péptidos , Familia de Multigenes , Vías Biosintéticas/genéticaRESUMEN
It is difficult for beginners to learn and use amplicon analysis software because there are so many software tools to choose from, and all of them need multiple steps of operation. Herein, we provide a cross-platform, open-source, and community-supported analysis pipeline EasyAmplicon. EasyAmplicon has most of the modules needed for an amplicon analysis, including data quality control, merging of paired-end reads, dereplication, clustering or denoising, chimera detection, generation of feature tables, taxonomic diversity analysis, compositional analysis, biomarker discovery, and publication-quality visualization. EasyAmplicon includes more than 30 cross-platform modules and R packages commonly used in the field. All steps of the pipeline are integrated into RStudio, which reduces learning costs, keeps the flexibility of the analysis process, and facilitates personalized analysis. The pipeline is maintained and updated by the authors and editors of WeChat official account "Meta-genome." Our team will regularly release the latest tutorials both in Chinese and English, read the feedback from users, and provide help to them in the WeChat account and GitHub. The pipeline can be deployed on various platforms, and the installation time is less than half an hour. On an ordinary laptop, the whole analysis process for dozens of samples can be completed within 3 h. The pipeline is available at GitHub (https://github.com/YongxinLiu/EasyAmplicon) and Gitee (https://gitee.com/YongxinLiu/EasyAmplicon).
RESUMEN
There are six major types of CRISPR-Cas systems that provide adaptive immunity in bacteria and archaea against invasive genetic elements. The discovery of CRISPR-Cas systems has revolutionized the field of genetics in many organisms. In the past few years, exploitations of the most abundant class 1 type I CRISPR-Cas systems have revealed their great potential and distinct advantages to achieve gene editing and regulation in diverse microorganisms in spite of their complicated structures. The widespread and diversified type I CRISPR-Cas systems are becoming increasingly attractive for the development of new biotechnological tools, especially in genetically recalcitrant microbial strains. In this review article, we comprehensively summarize recent advancements in microbial gene editing and regulation by utilizing type I CRISPR-Cas systems. Importantly, to expand the microbial host range of type I CRISPR-Cas-based applications, these structurally complicated systems have been improved as transferable gene-editing tools with efficient delivery methods for stable expression of CRISPR-Cas elements, as well as convenient gene-regulation tools with the prevention of DNA cleavage by obviating deletion or mutation of the Cas3 nuclease. We envision that type I CRISPR-Cas systems will largely expand the biotechnological toolbox for microbes with medical, environmental and industrial importance.
RESUMEN
The prevalence and propagation of antimicrobial resistance (AMR) are serious global public health concerns. The large and the ever-increasing use of antibiotics in livestock is also considered a great concern. The extent of the similarity of acquired antibiotic resistance genes (ARGs) between humans and food animals and the driving factors underlying AMR transfer between them are not clear, although a link between ARGs in both hosts was proposed. To address this question, with swine and chicken as examples of food animals, we analyzed over 1,000 gut metagenomes of humans and food animals from over the world. A relatively high abundance and diversity of ARGs were observed in swine compared with those in humans as a whole. Commensal bacteria, particularly species from Clostridiales, contribute the most ARGs associated with mobile genetic elements (MGEs) and were found in both humans and food animals. Further studies demonstrate that overrepresented MGEs, namely, Tn4451/Tn4453 and TnAs3, are attributed mainly to the sharing between humans and food animals. A member of large resolvase family site-specific recombinases, TnpX, is found in Tn4451/Tn4453 which facilitates the insertions of the transient circular molecule. Although the variance in the transferability of ARGs in humans is higher than that in swine, a higher average transferability was observed in swine than that in humans. In conclusion, the potential antibiotic resistance hot spots with higher transferability in food animals observed in the present study emphasize the importance of surveillance for emerging resistance threats before they spread. IMPORTANCE Antimicrobial resistance (AMR) has proven to be a global public health concern. To conquer this increasingly worrying trend, an overarching, One Health approach has been used that brings together different sectors, but the fundamental knowledge of the relationship between humans, food animals, and their environments is not mature yet or is lacking in some aspect. With swine and chicken as examples of food animals, a large global data set of over 1,000 human and food animal gut metagenomes was analyzed with a focus on acquired antibiotic resistance genes (ARGs) associated with mobile genetic elements (MGEs) to answer this question. Outputs from this work open a new avenue to further our understanding of ARG transferability in food animals. It is a necessary milestone to better equip governmental agencies to monitor and pre-empt antibiotic resistance hot spots. This work will assist and give guidance on how to decipher other links within any One Health initiatives with expected positive feedback to human health.
Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana , Humanos , Animales , Porcinos/genética , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Bacterias/genética , Genes Bacterianos , MetagenomaRESUMEN
Pseudomonas aeruginosa can cause various types of infections and is one of the most ubiquitous antibiotic-resistant pathogens found in healthcare settings. It is capable of adapting to adverse conditions by transforming its motile lifestyle to a sessile biofilm lifestyle, which induces a steady state of chronic infection. However, mechanisms triggering the lifestyle transition of P. aeruginosa strains with clinical significance are not very clear. In this study, we reported a recently isolated uropathogenic hyper-biofilm producer PA_HN002 and characterized its genome to explore genetic factors that may promote its transition into the biofilm lifestyle. We first showed that high intracellular c-di-GMP content in PA_HN002 gave rise to its attenuated motilities and extraordinary strong biofilm. Reducing the intracellular c-di-GMP content by overexpressing phosphodiesterases (PDEs) such as BifA or W909_14950 converted the biofilm and motility phenotypes. Whole genome sequencing and comprehensive analysis of all the c-di-GMP metabolizing enzymes led to the identification of multiple mutations within PDEs. Gene expression assays further indicated that the shifted expression profile of c-di-GMP metabolizing enzymes in PA_HN002 might mainly contribute to its elevated production of intracellular c-di-GMP and enhanced biofilm formation. Moreover, mobile genetic elements which might interfere the endogenous regulatory network of c-di-GMP metabolism in PA_HN002 were analyzed. This study showed a reprogrammed expression profile of c-di-GMP metabolizing enzymes which may promote the pathoadaption of clinical P. aeruginosa into biofilm producers.
Asunto(s)
Proteínas de Escherichia coli , Pseudomonas aeruginosa , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Hidrolasas Diéster Fosfóricas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMEN
INTRODUCTION: The emergence of multidrug resistance in Bacteroides fragilis, especially the phylogenetic lineage carrying the carbapenemase gene cfiA, represents an increasing threat to human health. However, knowledge on the diversity of the multidrug-resistant strains and the genetic elements carrying the antibiotic resistance genes (ARGs) remains limited. AIM: The objective of the study was to describe the resistome in cfiA-positive B. fragilis. METHODS: A collection of cfiA-positive B. fragilis from diverse human (8 bacteremias, 15 wound infections) and animal (2 chickens, 2 pigs, 6 dogs, 3 cats) sources in Hong Kong, 2015-2017 was analysed by whole genome sequencing. RESULTS: In the 36 isolates, 13 distinct ARGs (total number 83, median 2, range 0-7 per isolate) other than cfiA were detected. ARGs encoding resistance to aminoglycosides, ß-lactams, macrolides, sulphonamides and tetracyclines were carried by CTn341-like, CTnHyb-like, Tn5220-like, Tn4555-like and Tn613-like transposons and were detected in phylogenetically diverse isolates of different host sources. Only few ARGs encoding resistance to metronidazole and tetracyclines were localized on plasmids. In two chicken isolates, a novel transposon (designated as Tn6994) was found to be involved in the dissemination of multiple ARGs mediating resistance to multiple antibiotics, including metronidazole and linezolid that are critically important for treatment of anaerobic infections. In mating experiments, Tn6994 and the associated phenotypic resistance could be transferred to Bacteroides nordii recipient. CONCLUSION: This study illustrates the importance of transposons in the dissemination of ARGs in the cfiA-positive division of B. fragilis. One Health approach is necessary to track the dissemination of ARGs.
Asunto(s)
Infecciones Bacterianas , Infecciones por Bacteroides , Aminoglicósidos , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Bacteroides fragilis/genética , Pollos , Perros , Farmacorresistencia Microbiana , Humanos , Linezolid , Macrólidos , Metronidazol , Pruebas de Sensibilidad Microbiana , Filogenia , Sulfonamidas , Porcinos , Tetraciclinas , Secuenciación Completa del Genoma , beta-Lactamasas/genética , beta-LactamasRESUMEN
OBJECTIVES: To compare the phylogeny of cfiA-positive Bacteroides fragilis isolates from diverse human and animal sources. METHOD: Complete genome sequences were obtained from 42 cfiA-positive B. fragilis isolates (Hong Kong, 2015-2017) and additional 24 genomes deposited in the GenBank (multiple countries, 1985-2019) were included. The genomic clusters were constructed using PopPUNK. The CfiA alleles and polymorphism in the cfiA locus were analyzed in silico. RESULTS: The 66 isolates were grouped into 12 genomic clusters (BFSC-1 to 12). Human infection isolates were distributed in diverse clusters, being many of them common to fecal isolates from both human and animals. Thirteen CfiA alleles including 2 novel ones were identified. CfiA-1 (n = 28) is the predominating allele, following by CfiA-13 (n = 8), CfiA-4 (n = 7) and CfiA-14 (n = 6). The other CfiA alleles were identified in 1-3 isolates. Six patterns of gene context were identified in the regions flanking cfiA locus. No consistent association between genomic clusters and CfiA alleles could be detected. Similarly, markedly elevated imipenem MIC was linked to the integration, immediately upstram of cfiA of an IS element but not the CfiA allele or gene context. CONCLUSION: The phylogeny of cfiA-positive B. fragilis isolates causing human diseases was diverse and overlaped with those from human and animal carriage.
Asunto(s)
Infecciones Bacterianas , Infecciones por Bacteroides , Alelos , Animales , Antibacterianos , Proteínas Bacterianas/genética , Bacteroides fragilis/genética , Genómica , Humanos , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/genéticaRESUMEN
Pneumococcal conjugate vaccines (PCVs) successfully decreased the incidence of invasive pneumococcal disease in children. However, many countries have reported serotype replacement and a rebound in diseases from non-vaccine serotypes. Here, we report the genomic investigation of a Streptococcus pneumoniae strain M215 that caused severe meningoencephalitis in an infant in 2019. The strain was assigned to serotype 24F using the bioinformatic pipeline SeroBA and pneumococcal type specific anti-sera. The strain was resistant to cotrimoxazole from mutations in both folA and folP genes. It was susceptible to penicillin and other non-ß-lactam antibiotics. Phylogenetically, it belongs to Global Pneumococcal Sequence Cluster (GPSC) 6 and multi-locus sequence type 162. A total of 38 virulence genes were detected in the genome of M215. Upon comparison of the profile of virulence genes, GPSC6 but not non-GPSC6 strains of serotype 24F and related serotypes were found to possess the major virulence determinant, pilus islet-1, comprising genes encoding sortases (srtB, srtC, srtD), pilus proteins (rrgA, rrgB and rrgC) and one transcriptional regulator (rlrA), which was previously described to be characteristic feature of international clones in the pre-PCV era. In our locality, this represented the first detection of serotype 24F and GPSC6/ST162 causing serious pneumococcal disease. The emergence of the non-vaccine serotype 24F GPSC6/ST162 lineage with molecular feature of high virulence is concerning and emphasizes the need for full characterization of strains causing severe disease.
Asunto(s)
Meningoencefalitis , Streptococcus pneumoniae , Niño , Genómica , Hong Kong , Humanos , Serogrupo , Streptococcus pneumoniae/genéticaRESUMEN
The emergence of antimicrobial-resistant (AMR) bacteria has become one of the most serious threats to global health, necessitating the development of novel antimicrobial strategies. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system, known as a bacterial adaptive immune system, can be repurposed to selectively target and destruct bacterial genomes other than invasive genetic elements. Thus, the CRISPR-Cas system offers an attractive option for the development of the next-generation antimicrobials to combat infectious diseases especially those caused by AMR pathogens. However, the application of CRISPR-Cas antimicrobials remains at a very preliminary stage and numerous obstacles await to be solved. In this mini-review, we summarize the development of using type I, type II, and type VI CRISPR-Cas antimicrobials to eradicate AMR pathogens and plasmids in the past a few years. We also discuss the most common challenges in applying CRISPR-Cas antimicrobials and potential solutions to overcome them.
RESUMEN
The Class 1 type I CRISPR-Cas systems represent the most abundant and diverse CRISPR systems in nature. However, their applications for generic genome editing have been hindered due to difficulties of introducing the class-specific, multi-component effectors (Cascade) in heterologous hosts for functioning. Here we established a transferrable Cascade system that enables stable integration and expression of a highly active type I-F Cascade in heterologous bacterial hosts for various genetic exploitations. Using the genetically recalcitrant Pseudomonas species as a paradigm, we show that the transferred Cascade displayed substantially higher DNA interference activity and greater editing capacity than both the integrative and plasmid-borne Cas9 systems, and enabled deletion of large fragments such as the 21-kb integrated cassette with efficiency and simplicity. An advanced I-F-λred system was further developed to enable editing in genotypes with poor homologous recombination capacity, clinical isolates lacking sequence information, and cells containing anti-CRISPR elements Acrs. Lastly, an 'all-in-one' I-F Cascade-mediated CRISPRi platform was developed for transcription modulation by simultaneous introduction of the Cascade and the programmed mini-CRISPR array in one-step. This study provides a framework for expanding the diverse type I Cascades for widespread, heterologous genome editing and establishment of editing techniques in 'non-model' bacterial species.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Pseudomonas/genética , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Recombinación Genética , Transcripción GenéticaRESUMEN
BACKGROUND: The spread of antibiotic resistance has become one of the most urgent threats to global health, which is estimated to cause 700,000 deaths each year globally. Its surrogates, antibiotic resistance genes (ARGs), are highly transmittable between food, water, animal, and human to mitigate the efficacy of antibiotics. Accurately identifying ARGs is thus an indispensable step to understanding the ecology, and transmission of ARGs between environmental and human-associated reservoirs. Unfortunately, the previous computational methods for identifying ARGs are mostly based on sequence alignment, which cannot identify novel ARGs, and their applications are limited by currently incomplete knowledge about ARGs. RESULTS: Here, we propose an end-to-end Hierarchical Multi-task Deep learning framework for ARG annotation (HMD-ARG). Taking raw sequence encoding as input, HMD-ARG can identify, without querying against existing sequence databases, multiple ARG properties simultaneously, including if the input protein sequence is an ARG, and if so, what antibiotic family it is resistant to, what resistant mechanism the ARG takes, and if the ARG is an intrinsic one or acquired one. In addition, if the predicted antibiotic family is beta-lactamase, HMD-ARG further predicts the subclass of beta-lactamase that the ARG is resistant to. Comprehensive experiments, including cross-fold validation, third-party dataset validation in human gut microbiota, wet-experimental functional validation, and structural investigation of predicted conserved sites, demonstrate not only the superior performance of our method over the state-of-art methods, but also the effectiveness and robustness of the proposed method. CONCLUSIONS: We propose a hierarchical multi-task method, HMD-ARG, which is based on deep learning and can provide detailed annotations of ARGs from three important aspects: resistant antibiotic class, resistant mechanism, and gene mobility. We believe that HMD-ARG can serve as a powerful tool to identify antibiotic resistance genes and, therefore mitigate their global threat. Our method and the constructed database are available at http://www.cbrc.kaust.edu.sa/HMDARG/ . Video abstract (MP4 50984 kb).