Use Of Shotgun Metagenomic Sequencing To Determine How The Human Gut Microbiome And Antibiotic Resistome Influence The Risk Of Recurrent Clostridioides Difficile Infection

Clostridioides difficile is an opportunistic enteric pathogen that can cause a range of symptoms in humans from diarrhea to pseudomembranous colitis. The risk of recurrence of C. difficile infection after initial treatment with antibiotics is high. Although prior studies have sought to understand the link between the human gut microbiome and the risk of recurrence, none have utilized shotgun metagenomic sequencing methods to establish a relationship between microbiota diversity and recurrence risk. In this study, stool samples were obtained from 47 patients at Yale New Haven Hospital who tested positive for an incident C. difficile infection. Shotgun metagenomic sequencing was used to characterize the abundance and diversity of the microbiota and the antibiotic resistome of each sample. The association between taxonomic diversity of the gastrointestinal microbiota and the risk for recurrence, case definition, and patient characteristics was analyzed. There was a significant association between age and the risk for recurrent infection, with older patients more likely to experience recurrence. Linear discriminant analysis effect size revealed that certain taxonomic groups were differentially prevalent in patients with and without a recurrent episode and in patients with community-acquired infections vs. those with hospital- or healthcare-associated infections. Those who experienced a recurrent infection had a higher abundance of Blautia producta, and a lower of abundance of Gardnerella vaginalis and Eggerthella lenta than patients who did not experience a recurrence. The microbiota of patients who had a community-acquired infection were more likely to include protective bacterial species and bacteriophage, such as Bacteroides, Faecalibacterium prausnitzii, and crAssphage. This study provides a preliminary exploration into the association between the human microbiome and the risk of C. difficile recurrence and demonstrates the value of using shotgun metagenomic sequencing for further investigation

Antimicrobial Resistance And Molecular Epidemiology Of Escherichia Coli Isolated From Urban And Rural River Systems

Antimicrobial resistance (AMR) is an emerging public health issue that threatens the efficacy of antibiotic treatment for bacterial infections and human health. Sources of antimicrobial resistance genes (ARGs) in the environment include wastewater treatment plants and animal feeding operations that discharge waste into waterways, such as rivers and streams. This retrospective descriptive study describes the presence of AMR, and specific ARGs in Escherichia coli isolates from two distinct watersheds, rural and urban, with the use of antimicrobial susceptibility testing, whole-genome sequencing (WGS) to detect ARGs, and multi-locus sequence typing. Antimicrobial susceptibility testing was performed for 143 E. coli isolates, 73 originating from a rural watershed and 70 originating from an urban watershed. E. coli isolates from the rural watershed had a significantly higher prevalence of phenotypic non-susceptibility and ARGs for tetracycline (21.9% vs. 2.9%, p \u3c 0.01) when compared to urban watershed isolates. Based on phenotypic-susceptibility testing, WGS data of 68 isolates were annotated for ARGs. These data were used for the prediction of antimicrobial susceptibility, demonstrating high accuracy for the prediction of non-susceptibility for tetracycline, trimethoprim-sulfamethoxazole, and cephalosporins. WGS multi-locus sequence typing (MLST) yielded 47 sequence types, dominated by ST58 (n=6), ST10 (n=5), and ST155 (n=4). Waterways are important reservoirs and disseminators of antimicrobial-resistant bacteria (ARB) and ARGs. The evaluation and monitoring of AMR and ARGs in aquatic environments will lead to improved health through better prevention and control of E. coli infections

Monitoring antibiotic resistance genes in wastewater environments: The challenges of filling a gap in the One-Health cycle

Antibiotic resistance (AR) is a global problem requiring international cooperation and coordinated action. Global monitoring must rely on methods available and comparable across nations to quantify AR occurrence and identify sources and reservoirs, as well as paths of AR dissemination. Numerous analytical tools that are gaining relevance in microbiology, have the potential to be applied to AR research. This review summarizes the state of the art of AR monitoring methods, considering distinct needs, objectives and available resources. Based on the overview of distinct approaches that are used or can be adapted to monitor AR, it is discussed the potential to establish reliable and useful monitoring schemes that can be implemented in distinct contexts. This discussion places the environmental monitoring within the One-Health approach, where two types of risk, dissemination across distinct environmental compartments, and transmission to humans, must be considered. The plethora of methodological approaches to monitor AR and the variable features of the monitored sites challenge the capacity of the scientific community and policy makers to reach a common understanding. However, the dialogue between different methods and the production of action-oriented data is a priority. The review aims to warm up this discussion

Screening of pathogenic microbiota harbouring antibiotic resistance genes from healthcare wastes in Malaysia : a high-throughput amplicon sequencing approach

The disposal of healthcare waste without prior elimination of pathogens and hazardous contaminants has negative effects on the environment and public health. In past research the microbiological assessment of healthcare wastes employed a culture approach that resulted in the identification of Bacillus sp. in a sample of treated solid healthcare wastes. The effectiveness of microwave in hazardous waste treatment studied based on the survival of tested microorganisms using the culture method may overlook the presence of other pathogens after treatment. Yet, there is scarce data reported on the complete microbial community in microwave-treated healthcare waste using next-generation sequencing technology. This study aimed to profile the complete microbial community, identify viable antibiotic-resistant bacteria in microwave-treated healthcare wastes collected from three different waste operators (FC, FV, and FA) in Peninsular Malaysia, and characterize pathogenic gene markers in isolated organisms. The samples were subjected to bacterial and fungal amplicon sequencing for microbial community characterization, by targeting the full-length 16S ribosomal RNA (rRNA) gene and partial 18S rRNA gene with full-length internal transcribed spacer (ITS) 1 and ITS 2 regions, respectively. Bacterial cultivation was performed to identify viable bacteria in healthcare wastes. The isolated antibiotic-resistant bacteria were subjected to species identification and whole genome sequencing for complete genome characterization. In addition, antibiotic susceptibility testing was performed on the confirmed isolates using the disk diffusion technique to determine the antibiotic resistance patterns. Based on the results of objective 1, the bacterial composition in FC samples was dominated by the Aerococcus, Comamonas, and Pseudomonas genera, while FV and FA were dominated by Bacillus, Paenibacillus, and unclassified Bacilli. All three sets of samples showed significant differences in bacterial diversity, as evidenced by the alpha- (p-value = 0.048) and beta-diversity (p-value < 0.006) analyses. The fungal composition differed significantly between three groups of samples, as evidenced by the alpha- (p-value = 0.045) and beta-diversity (p-value < 0.002). The deep bioinformatic analysis confirmed the presence of blaTEM-1 and penP, which are associated with the production of class A beta-lactamase and beta-lactam resistance pathways. Based on objective 2, the viable bacteria in VFC, VFV, and VFA samples were represented by Proteus, Stenotrophomonas, and Pseudomonas genera, respectively, with significant beta diversity (p-value = 0.003). Based on the BLASTN results, the primary antibiotic-resistant bacteria isolated from VFC, VFV, and VFA samples were Proteus mirabilis, Stenotrophomonas maltophilia, and Pseudomonas sp., respectively. As no specific Pseudomonas species were identified from the database, this bacterium is potentially present as a novel bacterium. For objective 3, P. mirabilis and S. maltophilia were discovered to contain genes associated with virulence function and transposable element expression. The antibiotic resistance genes blaOXA-10 and sul1 were identified in P. mirabilis and S. maltophilia, conferring resistance to beta-lactam and folate pathway antagonist antibiotics. The antibiotic susceptibility tests revealed that P. mirabilis and S. maltophilia were multidrug-resistant bacteria, exhibiting resistance to drugs from multiple classes, including carbapenem. In conclusion, microorganisms and contaminants, which serve as putative indicators in healthcare waste treatment evaluation, revealed the limitations of the microwave sterilization method in microbial inactivation. Our findings suggested that the occurrence of clinically relevant microorganisms, antibiotic contaminants, and associated antibiotic resistance genes represents environmental and human health hazards when released into landfills via horizontal gene transfer

Antibiootikumiresistentsus omavahel seotud tehislikus ja looduslikus veekeskkonnas

Väitekirja elektrooniline versioon ei sisalda publikatsiooneAntibiootikumid on kõige olulisemad bakteriaalsete infektsioonide ravimiseks mõeldud ained, kuid kõikide antibiootikumide vastu tekib bakteritel ühel hetkel resistentsus, st et antibiootikum ei suuda enam bakterit tappa. Nii on antibiootikumiresistentsusest kujunenud tänapäeval üks suurimaid tervishoiuprobleeme maailmas. See ei ole siiski ainult meditsiinisektori probleem, vaid selle tekke ja leviku taga on ka antibiootikumide kasutamine põllumajanduses ja loomakasvatuses. Reoveepuhastusjaamade heitvesi on üks peamisi teid, kuidas resistentsus tehiskeskkonnast looduslikku keskkonda pääseb, kuna reoveepuhastuse käigus ei eemaldata kõiki antibiootikumijääke, resistentseid baktereid ega ka antibiootikumiresistentsust põhjustavaid geene. Kuna reoveepuhastusjaamade heitvesi juhitakse enamasti looduslikesse veekogudesse, näiteks ojadesse või jõgedesse, suureneb resistentsusnäitajate arv reoveepuhasti väljavoolust allavoolu jäävatel aladel. Antibiootikumiresistentsed bakterid ja resistentsusgeenid võivad kanduda edasi põhjavette, jõgedesse ja lõpuks ka merre. Sealt võivad need omakorda inimesele tagasi kanduda, näiteks saastunud vett juues, mereande süües või ujudes. Siinse doktoritöö eesmärgiks oli kirjeldada, kuidas reoveepuhastusjaamast pärinevad antibiootikumiresistentsusgeenid levivad allavoolu jäävasse ojja, sealt edasi jõkke ning viimaks Läänemerre. Töö tulemustest selgus, et reoveepuhastusjaamal on kõige suurem mõju vahetus läheduses (0,3 kilomeetrit) oleva veekeskkonna mikroobikoosluse struktuurile ja antibiootikumiresistentsusgeenide arvukusele. Samas juba 3,7 kilomeetrit eemal oli jõe bakterikooslus võrreldav reoveepuhastist ülesvoolu jääva alaga. Siiski tulenevalt Läänemere eripäradest – suur reostuskoormus, tihe rannikuala asustatus, suur valgala, aeglane veevahetus, madal vesi – on merekeskkond väga tundlik antibiootikumiresistentsusgeenide reostuse suhtes.Antibiotic resistance in connected engineered and natural aquatic environments Antibiotics are the most important drugs for treating bacterial infections, but all antibiotics introduced are susceptible to resistance, which means that the antibiotic no longer kills the bacteria. Thus, antibiotic resistance has become one of the biggest human health problems in the world today. Antibiotic resistance is not only a problem in medicine, but the use of antibiotics in agriculture and animal husbandry also play an important role in the spread of antibiotic resistance. Wastewater treatment plants effluent is one of the main pathways by which the resistance determinants from the human environment are introduced to the natural environment, because not all antibiotic residues, resistant bacteria and antibiotic resistance encoding genes are removed during wastewater treatment process. Since wastewater treatment plants effluent is often discharged into natural waterbodies, such as streams and rivers, the number of resistance determinants in downstream water increases. Antibiotic resistant bacteria and resistance genes can be transmitted to groundwater, rivers and ultimately to the sea, from where they can also be transmitted back to humans - for example, by drinking contaminated water, eating seafood or swimming. The aim of this thesis was to describe the dissemination pathway of the antibiotic resistance genes originating from the wastewater treatment plant effluent through the primary receiving waterbody to the final receiving waterbody (Baltic Sea). The results indicated that the wastewater treatment plant has the greatest impact on the aquatic environment in the close vicinity (0.3 kilometers) of the effluent as already 3.7 kilometers downstream the bacterial community of the river was comparable to the area upstream of the wastewater treatment plant effluent. However, the Baltic Sea is very vulnerable to ARG contamination due to its specifics (long water retention time, shallowness, large catchment area, high pollution load).https://www.ester.ee/record=b546564

Community-based Analysis for Identifying Populations Relevant to Pollutant Mitigation in Natural and Engineered Processes

Microorganisms are involved in various important environmental processes. While current understanding of these microbial processes is shaped to a large extent by studies of individual populations, increasing efforts have been made to understanding the roles of microbial communities as a whole in environmental processes, which is made possible with the development of high-throughput sequencing technologies. In this dissertation, the microbial communities in anaerobic waste treatment processes and stream waters influenced by anthropogenic activities were investigated as models of engineered and natural systems using microbial community-based analyses. In the anaerobic waste treatment processes, metagenomics analyses revealed the persistence of antibiotic resistant genes (ARGs) and association with specific microbial hosts, providing insight into potential targets for mitigating the spread of ARGs. Further, community-based analysis identified legacy effect as an important mechanism contributing to the assembly of microbial communities, shedding light on potential strategies for the control of important populations underlying waste treatment. In the investigation of stream waters impacted by anthropogenic activities, microbial community-based analyses enabled the successful identification of primary anthropogenic sources contributing to the microbial contamination in stream water, which has long been confounded using traditional indicator-based approaches. Results from this study provide an innovative technique for microbial source tracking not otherwise possible with individual population-based approaches. Community-based analyses, as demonstrated in this dissertation, are capable of identifying interactions between microbial populations which are essential for the survival, persistence, and function of microorganisms in the environment. Furthermore, community-based analyses are capable of utilizing all information embedded in microbial communities, which enables more precise and accurate quantification of microbial community composition and function, paving the way for the development of more effective data analytics techniques for the characterization and modeling of microbial communities

CHARACTERIZATION OF THE ENVIRONMENTAL RESISTOME IN THE GALAPAGOS ISLANDS, ECUADOR: A ONE HEALTH PERSPECTIVE

Antibiotic resistance represents one of our generation’s most pressing public health challenges, with some exports warning of an approaching post-antibiotic era. Mitigating this threat requires an understanding of the evolutionary ecology of resistance, including the unique ability of microorganisms to move between humans, animals, and the environment. However, significant questions remain regarding the role of the environment as a source and reservoir for antibiotic resistance. Moreover, there few environments left on earth where we can study background antibiotic resistance in the absence of significant anthropogenic influence. The Galapagos Islands of Ecuador, where the human population is restricted to 3% of the landmass, represent a unique model system to study how human activity influences antibiotic resistance patterns in wildlife and the environment in a largely protected ecosystem. With samples from humans, animals, and the environment, we designed a One Health study aimed at answering what, where, and who: what antibiotic resistance genes are present, where are they located in regards to mobile genetic elements, and who may be the presumptive bacterial host? We employed shotgun metagenomic sequencing to achieve a broad characterization of 90 environmental, wildlife, and human resistomes and mobilomes, and paired this data with targeted detection of the class I integron-integrase gene using a novel ddPCR assay in > 250 Galapagos samples. Additionally, we used a combination of 16S rRNA amplicon sequencing and taxonomic inference from metagenomes to profile the microbial communities associated with these samples. Our results suggest that human, environmental, and wildlife reservoirs are characterized by distinct resistomes and mobilomes, with overall abundance and diversity of antibiotic resistance genes (ARGs) increasing along a gradient of anthropogenic influence. Overall, we found wildlife to harbor fewer ARGs than wastewater and humans, though some exceptions were noted among land iguanas. Differential abundance analysis revealed ARGs unique to each wildlife species with possible bacterial hosts identified in taxonomic assignments in some cases. We recorded overall agreement between resistome and mobilome data sets, and correlation between taxa, ARGs, and MGEs pointed to a key relationship with Enterobacteriaceae.Doctor of Philosoph

Characterising antibiotic susceptibility and resistance in human commensal gut bacteria

Abstract Student name: Lindsay Pike Thesis title: Characterising antibiotic susceptibility and resistance in human commensal gut bacteria The human commensal gut microbiota can act as a reservoir of antimicrobial resistance genes that can persist and spread to pathogens. However, the extent and diversity of antibiotic resistance encoded by human commensal bacteria remains to be determined. Due to immediate clinical relevance and our previous inability to culture these commensal bacteria, the majority of research into antibiotic resistance has focused on pathogenic organisms or well-characterized antibiotic resistance mechanisms. Here, I demonstrate the existence of unpredicted antibiotic resistance, not detected by several genome-based prediction methods, in diverse bacterial species from the human gastrointestinal tract. 178 antibiotic resistance genes and mutations were identified in a culture collection of 737 phylogenetically diverse gut bacteria from healthy humans. Recent developments in culturing anaerobic gut bacteria were used to determine antibiotic sensitivity phenotypes and observe the spectrum of clinically relevant antibiotics across the diversity of these isolates. These data were combined to assess the accuracy of genome-based predictions in human commensal gut bacteria, revealing multiple instances of unpredicted antibiotic resistance. This highlights the importance of combining computational genomic prediction with functional validation and increases our knowledge of antibiotic resistance in commensal human gut bacteria. In addition, the impact of therapeutic amoxicillin treatment on antibiotic resistance in mice with human-derived gut microbiota was studied. These experiments model processes in humans and reveal community- and strain-level changes in antibiotic resistance following antibiotic therapy. These experiments further elucidate the role of the gut microbiota as a reservoir of antibiotic resistance and the influence of antibiotics on this reservoir.My PhD was funded by the Medical Research Council (MRC) and the Wellcome Sanger Institute