RESUMEN
One Health surveillance involves the analysis of human, animal and environmental samples, recognising their interconnectedness in health systems. Such considerations are crucial to investigate the transmission of many pathogens, including drug-resistant bacteria and parasites. The highest rates of antimicrobial resistance (AMR)-associated deaths are observed in sub-Saharan Africa, where concurrently the waterborne parasitic disease schistosomiasis can be highly endemic in both humans and animals. Although there is growing acknowledgment of significant interactions between bacteria and parasites, knowledge of relationships between schistosomes, microbes and AMR remains inadequate. In addition, newly emergent research has revealed the previously underappreciated roles of animals and the environment in both AMR and schistosomiasis transmission. We consider shared environmental drivers and colonisation linkage in this narrative review, with a focus on extended-spectrum beta-lactamase-mediated resistance among bacteria from the Enterobacteriaceae family, which is exceedingly prevalent and responsible for a high burden of AMR-associated deaths. Then we examine novel findings from Malawi, where the landscapes of AMR and schistosomiasis are rapidly evolving, and make comparisons to other geographic areas with similar co-infection epidemiology. We identify several knowledge gaps that could be addressed in future research, including the need to characterise the impact of intestinal schistosomiasis and freshwater contact on intestinal AMR colonisation, before proposing a rationale for connecting AMR surveillance and schistosomiasis research within a One Health framework.
Asunto(s)
Salud Única , Esquistosomiasis , Humanos , Esquistosomiasis/epidemiología , Esquistosomiasis/tratamiento farmacológico , Esquistosomiasis/transmisión , Animales , Malaui/epidemiología , Enterobacteriaceae/efectos de los fármacos , Schistosoma/efectos de los fármacos , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana , Infecciones por Enterobacteriaceae/epidemiologíaRESUMEN
Aim: Proof-of-concept study, highlighting the clinical diagnostic ability of FT-IR compared with MALDI-TOF MS, combined with WGS. Materials & methods: 104 pathogenic isolates of Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus were analyzed. Results: Overall prediction accuracy was 99.6% in FT-IR and 95.8% in MALDI-TOF-MS. Analysis of N. meningitidis serogroups was superior in FT-IR compared with MALDI-TOF-MS. Phylogenetic relationship of S. pyogenes was similar by FT-IR and WGS, but not S. aureus or S. pneumoniae. Clinical severity was associated with the zinc ABC transporter and DNA repair genes in S. pneumoniae and cell wall proteins (biofilm formation, antibiotic and complement permeability) in S. aureus via WGS. Conclusion: FT-IR warrants further clinical evaluation as a promising diagnostic tool.
We tested a technique (FT-IR) to identify four different, common bacteria from 104 children with serious infections and compared it to lab methods for diagnosis. FT-IR was more accurate. We tested if it could identify subtypes of bacteria, which is important in outbreaks. It was able to subtype two species, but not the two other species. However, it is a much faster and cheaper technique than the gold standard. It may be useful in certain outbreaks. We also investigated the trends between genes and the length of hospital stay. This can support further laboratory research. As a fast, low-cost test, FT-IR warrants further testing before it is applied to clinical labs.
Asunto(s)
Neisseria meningitidis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Streptococcus pneumoniae , Streptococcus pyogenes , Secuenciación Completa del Genoma , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Humanos , Neisseria meningitidis/genética , Neisseria meningitidis/clasificación , Neisseria meningitidis/aislamiento & purificación , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/aislamiento & purificación , Streptococcus pyogenes/genética , Streptococcus pyogenes/clasificación , Filogenia , Staphylococcus aureus/genética , Genoma Bacteriano/genética , Prueba de Estudio Conceptual , Bacterias/genética , Bacterias/clasificación , Bacterias/aislamiento & purificaciónRESUMEN
Antimicrobial resistance disseminates throughout bacterial populations via horizontal gene transfer, driven mainly by mobile genetic elements (MGEs). Entrapment vectors are key tools in determining MGE movement within a bacterial cell between different replicons or between sites within the same replicon. The pBACpAK entrapment vector has been previously used to study intracellular transfer in Gram-negative bacteria however since pBACpAK contains a chloramphenicol resistance gene, it cannot be used in bacterial isolates which are already resistant to chloramphenicol. Therefore, we developed new derivatives of the pBACpAK entrapment vector to determine intracellular transfer of MGEs in an Escherichia coli DH5α transconjugant containing the chloramphenicol resistance plasmid pD25466. The catA1 of pBACpAK was replaced by both mcr-1 in pBACpAK-COL and aph(3')-Ia in pBACpAK-KAN, allowing it to be used in chloramphenicol resistant strains. The plasmid constructs were verified and then used to transform the E. coli DH5α/pD25466 transconjugants in order to detect intracellular movement of the MGEs associated with the pD25466 plasmid. Here we report on the validation of the expanded suite of pBACpAK vectors which can be used to study the intracellular transfer of MGEs between, and within, replicons in bacteria with different antimicrobial resistance profiles.
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Cloranfenicol , Escherichia coli , Cloranfenicol/farmacología , Antibacterianos/farmacología , Plásmidos/genética , Secuencias Repetitivas Esparcidas , Farmacorresistencia Bacteriana/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
Here, we provide the genome sequence of a Leclercia adecarboxylata isolated from a screen of an environmental bacterial isolate library for resistance to the plant flavonoid berberine. We detected the colistin resistance gene mcr-9, located on an IncFII(pECLA) plasmid.
Asunto(s)
Genómica , beta-Lactamasas , Adulto , Humanos , Malaui/epidemiología , beta-Lactamasas/genéticaRESUMEN
BACKGROUND: Low-income countries have high morbidity and mortality from drug-resistant infections, especially from enteric bacteria such as Escherichia coli. In these settings, sanitation infrastructure is of variable and often inadequate quality, creating risks of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales transmission. We aimed to describe the prevalence, distribution, and risks of ESBL-producing Enterobacterales colonisation in sub-Saharan Africa using a One Health approach. METHODS: Between April 29, 2019, and Dec 3, 2020, we recruited 300 households in Malawi for this longitudinal cohort study: 100 each in urban, peri-urban, and rural settings. All households underwent a baseline visit and 195 were selected for longitudinal follow-up, comprising up to three additional visits over a 6 month period. Data on human health, antibiotic usage, health-seeking behaviours, structural and behavioural environmental health practices, and animal husbandry were captured alongside human, animal, and environmental samples. Microbiological processing determined the presence of ESBL-producing E coli and Klebsiella pneumoniae, and hierarchical logistic regression was performed to evaluate the risks of human ESBL-producing Enterobacterales colonisation. FINDINGS: A paucity of environmental health infrastructure and materials for safe sanitation was identified across all sites. A total of 11 975 samples were cultured, and ESBL-producing Enterobacterales were isolated from 1190 (41·8%) of 2845 samples of human stool, 290 (29·8%) of 973 samples of animal stool, 339 (66·2%) of 512 samples of river water, and 138 (46·0%) of 300 samples of drain water. Multivariable models illustrated that human ESBL-producing E coli colonisation was associated with the wet season (adjusted odds ratio 1·66, 95% credible interval 1·38-2·00), living in urban areas (2·01, 1·26-3·24), advanced age (1·14, 1·05-1·25), and living in households where animals were observed interacting with food (1·62, 1·17-2·28) or kept inside (1·58, 1·00-2·43). Human ESBL-producing K pneumoniae colonisation was associated with the wet season (2·12, 1·63-2·76). INTERPRETATION: There are extremely high levels of ESBL-producing Enterobacterales colonisation in humans and animals and extensive contamination of the wider environment in southern Malawi. Urbanisation and seasonality are key risks for ESBL-producing Enterobacterales colonisation, probably reflecting environmental drivers. Without adequate efforts to improve environmental health, ESBL-producing Enterobacterales transmission is likely to persist in this setting. FUNDING: Medical Research Council, National Institute for Health and Care Research, and Wellcome Trust. TRANSLATION: For the Chichewa translation of the abstract see Supplementary Materials section.
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Antiinfecciosos , Infecciones por Escherichia coli , Infecciones por Klebsiella , Salud Única , Animales , Humanos , Escherichia coli , Klebsiella pneumoniae , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/microbiología , Estudios Longitudinales , beta-Lactamasas , Infecciones por Klebsiella/epidemiología , Infecciones por Klebsiella/microbiología , Estudios de CohortesRESUMEN
BACKGROUND: Fluoroquinolones have been, and continue to be, routinely used for treatment of many bacterial infections. In recent years, most parts of the world have reported an increasing trend of fluoroquinolone resistant (FQR) Gram-negative bacteria. METHODS: A cross-sectional study was conducted between March 2017 and July 2018 among children admitted due to fever to referral hospitals in Dar es Salaam, Tanzania. Rectal swabs were used to screen for carriage of extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-PE). ESBL-PE isolates were tested for quinolone resistance by disk diffusion method. Randomly selected fluroquinolone resistant isolates were characterized by using whole genome sequencing. RESULTS: A total of 142 ESBL-PE archived isolates were tested for fluoroquinolone resistance. Overall phenotypic resistance to ciprofloxacin, levofloxacin and moxifloxacin was found in 68% (97/142). The highest resistance rate was seen among Citrobacter spp. (100%, 5/5), followed by Klebsiella. pneumoniae (76.1%; 35/46), Escherichia coli (65.6%; 42/64) and Enterobacter spp. (31.9%; 15/47). Whole genome sequencing (WGS) was performed on 42 fluoroquinolone resistant-ESBL producing isolates and revealed that 38/42; or 90.5%, of the isolates carried one or more plasmid mediated quinolone resistance (PMQR) genes. The most frequent PMQR genes were aac(6')-lb-cr (74%; 31/42), followed by qnrB1 (40%; 17/42), oqx, qnrB6 and qnS1. Chromosomal mutations in gyrA, parC and parE were detected among 19/42 isolates, and all were in E. coli. Most of the E. coli isolates (17/20) had high MIC values of > 32 µg/ml for fluoroquinolones. In these strains, multiple chromosomal mutations were detected, and all except three strains had additional PMQR genes. Sequence types, ST131 and ST617 predominated among E. coli isolates, while ST607 was more common out of 12 sequence types detected among the K. pneumoniae. Fluoroquinolone resistance genes were mostly associated with the IncF plasmids. CONCLUSION: The ESBL-PE isolates showed high rates of phenotypic resistance towards fluoroquinolones likely mediated by both chromosomal mutations and PMQR genes. Chromosomal mutations with or without the presence of PMQR were associated with high MIC values in these bacteria strains. We also found a diversity of PMQR genes, sequence types, virulence genes, and plasmid located antimicrobial resistance (AMR) genes towards other antimicrobial agents.
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Fluoroquinolonas , Quinolonas , Niño , Humanos , Fluoroquinolonas/farmacología , Tanzanía/epidemiología , Estudios Transversales , Escherichia coli/genética , Quinolonas/farmacologíaRESUMEN
Mobile colistin resistance (mcr) genes are often located on conjugative plasmids, where their association with insertion sequences enables intercellular and intracellular dissemination throughout bacterial replicons and populations. Multiple mcr genes have been discovered in every habitable continent, in many bacterial species, on both plasmids and integrated into the chromosome. Previously, we showed the intercellular transfer of mcr-1 on an IncI1 plasmid, pMCR-E2899, between strains of Escherichia coli. Characterizing the intracellular dynamics of mcr-1 transposition and recombination would further our understanding of how these important genes move through bacterial populations and whether interventions can be put in place to stop their spread. In this study, we aimed to characterize transfer events from the mcr-1-containing transposon Tn7511 (ISApl1-mcr-1-pap2-ISApl1), located on plasmid pMCR-E2899, using the pBACpAK entrapment vector. Following the transformation of pBACpAK into our DH5α-Azir/pMCR-E2899 transconjugant, we captured ISApl1 in pBACpAK multiple times and, for the first time, observed the ISApl1-mediated transfer of the mcr-1 transposon (Tn7511) into the chromosome of E. coli DH5α. Whole-genome sequencing allowed us to determine consensus insertion sites of ISApl1 and Tn7511 in this strain, and comparison of these sites allowed us to explain the transposition events observed. These observations reveal the consequences of ISApl1 transposition within and between multiple replicons of the same cell and show mcr-1 transposition within the cell as part of the novel transposon Tn7511. IMPORTANCE By analyzing the intracellular transfer of clinically relevant transposons, we can understand the dissemination and evolution of drug resistance conferring mobile genetic elements (MGEs) once a plasmid enters a cell following conjugation. This knowledge will help further our understanding of how these important genes move through bacterial populations. Utilizing the pBACpAK entrapment vector has allowed us to determine the mobility of the novel mcr-1-containing transposon Tn7511.
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Colistina , Proteínas de Escherichia coli , Colistina/farmacología , Antibacterianos/farmacología , Escherichia coli/genética , Farmacorresistencia Bacteriana/genética , Plásmidos/genética , Elementos Transponibles de ADN , Proteínas de Escherichia coli/genética , Bacterias/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
Antimicrobial resistance (AMR) is an increasingly important global challenge for healthcare systems as well as agricultural food production systems. Our ability to prepare for, and respond to, emerging AMR threats is dependent on our knowledge of genes able to confer AMR that are circulating within various environmental, animal, and human microbiomes. Targeted, sequence-specific, detection of AMR genes and functional resistance assays, described here, carried out on metagenomic DNA gives us unique insights into the presence of AMR genes and how these are associated with mobile genetic elements that may be responsible for their dissemination and can also provide important information about the mechanisms of resistance underpinning the phenotype.
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Antibacterianos , Antiinfecciosos , Animales , Humanos , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Metagenómica , Antiinfecciosos/farmacología , ADNRESUMEN
Emergence of novel human pathogens pose significant challenges to human health as highlighted by the SARS-CoV-2 pandemic. Wastewater based epidemiology (WBE) has previously been employed to identify viral pathogens and outbreaks by testing samples from regional wastewater treatment plants. Near source tracking (NST) allows for more targeted WBE by analysing samples from individual buildings such as schools or even individual floors such as in multi-floor office buildings. Despite the public health advantages of WBE, few strategies exist for optimising NST sampling methodologies. Therefore, we developed a protocol to evaluate virus detection in NST sampling using Pepper Mild Mottle Virus (PMMoV) as a proxy for RNA viruses. PMMoV is the most abundant enteric human associated RNA virus and is present in peppers/pepper-containing foods. Two bespoke TaqMan RT-PCR assays were developed to detect a PMMoV genomic 5' region and a capsid associated gene. To evaluate the protocol against field samples, pepper homogenates were flushed down an in-use toilet (Liverpool School of Tropical Medicine, UK) to spike wastewater with PMMoV on multiple days, and samples collected from two sewage access points to validate NST samplers. These wastewater samples were assessed for PMMoV based on Ct values and results compared to pepper and Tabasco derived PMMoV positive controls. Positive detection of PMMoV was comparable and consistent in ten independent samples across two NST samplers regardless of pepper homogenate spiking. We have developed two novel one step TaqMan assays that amplify both PMMoV targets in viral RNA extractions from peppers, Tabasco, and wastewater samples with cDNA synthesis through to RT-PCR results taking approximately 30 minutes. Pepper homogenate flushing was not required to detect PMMoV in our wastewater samples, however this strategy of flushing PMMoV containing materials outlined here could be valuable in assessing and validating NST in buildings with no previous or current sewage flow.
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COVID-19 , Tobamovirus , Humanos , Aguas Residuales , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Aguas del Alcantarillado , Monitoreo Epidemiológico Basado en Aguas Residuales , SARS-CoV-2/genética , Tobamovirus/genéticaRESUMEN
Resistance to piperacillin/tazobactam (TZP) in Escherichia coli has predominantly been associated with mechanisms that confer resistance to third-generation cephalosporins. Recent reports have identified E. coli strains with phenotypic resistance to piperacillin/tazobactam but susceptibility to third-generation cephalosporins (TZP-R/3GC-S). In this study we sought to determine the genetic diversity of this phenotype in E. coli (n=58) isolated between 2014-2017 at a single tertiary hospital in Liverpool, UK, as well as the associated resistance mechanisms. We compare our findings to a UK-wide collection of invasive E. coli isolates (n=1509) with publicly available phenotypic and genotypic data. These data sets included the TZP-R/3GC-S phenotype (n=68), and piperacillin/tazobactam and third-generation cephalosporin-susceptible (TZP-S/3GC-S, n=1271) phenotypes. The TZP-R/3GC-S phenotype was displayed in a broad range of sequence types, which was mirrored in the same phenotype from the UK-wide collection, and the overall diversity of invasive E. coli isolates. The TZP-R/3GC-S isolates contained a diverse range of plasmids, indicating multiple acquisition events of TZP resistance mechanisms rather than clonal expansion of a particular plasmid or sequence type. The putative resistance mechanisms were equally diverse, including hyperproduction of TEM-1, either via strong promoters or gene amplification, carriage of inhibitor-resistant ß-lactamases, and an S133G blaCTX-M-15 mutation detected for the first time in clinical isolates. Several of these mechanisms were present at a lower abundance in the TZP-S/3GC-S isolates from the UK-wide collection, but without the associated phenotypic resistance to TZP. Eleven (19%) of the isolates had no putative mechanism identified from the genomic data. Our findings highlight the complexity of this cryptic phenotype and the need for continued phenotypic monitoring, as well as further investigation to improve detection and prediction of the TZP-R/3GC-S phenotype from genomic data.
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Infecciones por Escherichia coli , Sepsis , Antibacterianos/farmacología , Cefalosporinas/farmacología , Escherichia coli/genética , Infecciones por Escherichia coli/epidemiología , Humanos , Combinación Piperacilina y TazobactamRESUMEN
OBJECTIVES: The COVID-19 pandemic has highlighted both the vulnerabilities and the critical role of global pharmaceutical systems in enabling equitable access to medicines. In this personal view, we position the pharmaceutical system as a missed research and investment opportunity that, if integrated properly, would benefit antimicrobial stewardship (AMS) programmes within a One Health approach. KEY FINDINGS: The pharmaceutical supply management cycle (PSMC) illustrates the continuous interdependence between four key phases: selection, procurement, distribution and use. Furthermore, a PSMC is subject to external forces of market competition, policy and regulation - across human, animal and environmental health. We present examples of overlap in PSMCs across different One Health sectors and discuss the need for integration within human, animal and environmental health contexts. SUMMARY: Despite pharmaceutical systems being fundamental to successful AMS programmes, they are currently neglected and undervalued. Research and investment into pharmaceutical system optimisation and integration into AMS programmes present an opportunity for both high-income countries and low- and middle-income countries to develop responsible, comparable and international AMS innovations and interventions.
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Antiinfecciosos , Programas de Optimización del Uso de los Antimicrobianos , COVID-19 , Salud Única , Animales , Antiinfecciosos/uso terapéutico , Humanos , Pandemias , Preparaciones FarmacéuticasRESUMEN
Introduction. Carbapenem-resistant members of the family Enterobacteriaceae are emerging as a global public-health threat and cause substantial challenges in clinical practice.Gap Statement. There is a need for increased and continued genomic surveillance of antimicrobial resistance genes globally in order to detect outbreaks and dissemination of clinically important resistance genes and their associated mobile genetic elements in human pathogens.Aim. To describe the resistance mechanisms of carbapenem-resistant Escherichia coli.Methods. Rectal swabs from neonates and newly diagnosed human immunodeficiency virus (HIV) infected adults were collected between April 2017 and May 2018 and screened for faecal carriage of carbapenamases and OXA-48 producing members of the family Enterobacteriaceae. Bacterial isolates were identified using matrix assisted laser desorption ionization time of flight mass spectrometry. Antimicrobial susceptibility testing was performed by E-test. Whole genomes of carbapenem-resistant E. coli were investigated using a hybrid assembly of Illumina and Oxford Nanopore Technologies sequencing reads.Results. Three carbapenem-resistant E. coli were detected, two from neonates and one from an HIV infected adult. All three isolates carried bla NDM-5. Two E. coli from neonates belonged to ST167 and bla NDM-5 co-existed with bla CTX-M-15 and bla OXA-01, and all were carried on IncFIA type plasmids. The E. coli from the HIV infected adult belonged to ST2083, and carried bla NDM-5 on an IncX3 type plasmid and bla CMY-42 on an IncI type plasmid. All bla NDM-5 carrying plasmids contained conjugation related genes. In addition, E. coli from the HIV infected adult carried three more plasmid types; IncFIA, IncFIB and Col(BS512). One E. coli from a neonate also carried one extra plasmid Col(BS512). All three E. coli harboured resistance genes to fluoroquinolone, aminoglycosides, sulfamethoxazole, trimethoprim, macrolides and tetracycline, carried on the IncFIA type plasmid. Furthermore, E. coli from the neonates carried a chloramphenicol resistance gene (catB3), also on the IncFIA plasmid. All three isolates were susceptible to colistin.Conclusion. This is the first report, to our knowledge, from Tanzania detecting bla NDM-5 producing E. coli. The carbapenemase gene was carried on an IncFIA and IncX3 type plasmids. Our findings highlight the urgent need for a robust antimicrobial resistance (AMR) surveillance system to monitor and rapidly report on the incidence and spread of emerging resistant bacteria in Tanzania.
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Infecciones por Escherichia coli , Escherichia coli , Infecciones por VIH , Adulto , Antibacterianos , Carbapenémicos , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/epidemiología , Infecciones por VIH/complicaciones , Humanos , Recién Nacido , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Tanzanía/epidemiología , beta-Lactamasas/clasificación , beta-Lactamasas/genéticaRESUMEN
Mobile genetic elements (MGEs) are often associated with antimicrobial resistance genes (ARGs). They are responsible for intracellular transposition between different replicons and intercellular conjugation and are therefore important agents of ARG dissemination. Detection and characterization of functional MGEs, especially in clinical isolates, would increase our understanding of the underlying pathways of transposition and recombination and allow us to determine interventional strategies to interrupt this process. Entrapment vectors can be used to capture active MGEs, as they contain a positive selection genetic system conferring a selectable phenotype upon the insertion of an MGE within certain regions of that system. Previously, we developed the pBACpAK entrapment vector that results in a tetracycline-resistant phenotype when MGEs translocate and disrupt the cI repressor gene. We have previously used pBACpAK to capture MGEs in clinical Escherichia coli isolates following transformation with pBACpAK. In this study, we aimed to extend the utilization of pBACpAK to other bacterial taxa. We utilized an MGE-free recipient E. coli strain containing pBACpAK to capture MGEs on conjugative, ARG-containing plasmids following conjugation from clinical Enterobacteriaceae donors. Following the conjugative transfer of multiple conjugative plasmids and screening for tetracycline resistance in these transconjugants, we captured several insertion sequence (IS) elements and novel transposons (Tn7350 and Tn7351) and detected the de novo formation of novel putative composite transposons where the pBACpAK-located tet(A) is flanked by ISKpn25 from the transferred conjugative plasmid, as well as the ISKpn14-mediated integration of an entire 119-kb, blaNDM-1-containing conjugative plasmid from Klebsiella pneumoniae. IMPORTANCE By analyzing transposition activity within our MGE-free recipient, we can gain insights into the interaction and evolution of multidrug resistance-conferring MGEs following conjugation, including the movement of multiple ISs, the formation of composite transposons, and cointegration and/or recombination between different replicons in the same cell. This combination of recipient and entrapment vector will allow fine-scale experimental studies of factors affecting intracellular transposition and MGE formation in and from ARG-encoding MGEs from multiple species of clinically relevant Enterobacteriaceae.
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Conjugación Genética , Elementos Transponibles de ADN , Farmacorresistencia Bacteriana Múltiple , Infecciones por Enterobacteriaceae/microbiología , Enterobacteriaceae/genética , Plásmidos/genética , Antibacterianos/farmacología , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Transferencia de Gen Horizontal , Humanos , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Plásmidos/metabolismoRESUMEN
In sub-Saharan Africa (sSA), there is high morbidity and mortality from severe bacterial infection and this is compounded by antimicrobial resistance, in particular, resistance to 3rd-generation cephalosporins. This resistance is typically mediated by extended-spectrum beta lactamases (ESBLs). To interrupt ESBL transmission it will be important to investigate how human behaviour, water, sanitation, and hygiene (WASH) practices, environmental contamination, and antibiotic usage in both urban and rural settings interact to contribute to transmission of ESBL E. coli and ESBL K. pneumoniae between humans, animals, and the environment. Here we present the protocol for the Drivers of Resistance in Uganda and Malawi (DRUM) Consortium, in which we will collect demographic, geospatial, clinical, animal husbandry and WASH data from a total of 400 households in Uganda and Malawi. Longitudinal human, animal and environmental sampling at each household will be used to isolate ESBL E. coli and ESBL K. pneumoniae. This will be complimented by a Risks, Attitudes, Norms, Abilities and Self-Regulation (RANAS) survey and structured observations to understand the contextual and psychosocial drivers of regional WASH practices. Bacterial isolates and plate sweeps will be further characterised using a mixture of short-,long-read and metagenomic whole-genome sequencing. These datasets will be integrated into agent-based models to describe the transmission of EBSL resistance in Uganda and Malawi and allow us to inform the design of interventions for interrupting transmission of ESBL-bacteria.
RESUMEN
A large proportion of bacteria, from a multitude of environments, are not yet able to be grown in the laboratory, and therefore microbiological and molecular biological investigations of these bacteria are challenging. A way to circumvent this challenge is to analyze the metagenome, the entire collection of DNA molecules that can be isolated from a particular environment or sample. This collection of DNA molecules can be sequenced and assembled to determine what is present and infer functional potential, or used as a PCR template to detect known target DNA and potentially unknown regions of DNA nearby those targets; however assigning functions to new or conserved hypothetical, functionally cryptic, genes is difficult. Functional metagenomics allows researchers to determine which genes are responsible for selectable phenotypes, such as resistance to antimicrobials and metabolic capabilities, without the prerequisite needs to grow the bacteria containing those genes or to already know which genes are of interest. It is estimated that a third of the resident species of the human oral cavity is not yet cultivable and, together with the ease of sample acquisition, makes this metagenome particularly suited to functional metagenomic studies. Here we describe the methodology related to the collection of saliva samples, extraction of metagenomic DNA, construction of metagenomic libraries, as well as the description of functional assays that have previously led to the identification of new genes conferring antimicrobial resistance.