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Efflux of antibiotics is an important survival strategy in bacteria. Mycobacterium tuberculosis has approximately sixty efflux pumps, but little is known about the role of each pump or the substrates they efflux. The putative efflux pump, EfpA, is a member of the major facilitator superfamily and has been shown to be essential by saturation transposon mutagenesis studies. It has been implicated in the efflux of isoniazid (INH), which is a first-line drug used to treat tuberculosis (TB). This is supported by evidence from transcriptional profiling showing that efpA is induced in response to INH exposure. However, its roles in the physiology and adaptation of M. tuberculosis to antibiotics have yet to be determined. In this study, we describe the repression of efpA in M. tuberculosis, using CRISPR interference (CRISPRi) to knockdown the expression of this essential gene and the direct effect of this on the ability of M. tuberculosis to survive exposure to INH over a 45-day time course. We determined that wild-type levels of efpA were required for recovery of M. tuberculosis following INH exposure and that, after 45 days of INH exposure, only a few viable colonies were recoverable from efpA-repressed M. tuberculosis. We conclude that EfpA is required for recovery of M. tuberculosis following INH exposure, which could reduce the efficacy of INH in vivo, and that EfpA may have a role in the development of resistance during drug therapy.
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Antituberculosos , Proteínas de Bactérias , Isoniazida , Mycobacterium tuberculosis , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade Microbiana , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacosRESUMO
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.
Assuntos
Saúde Única , Esquistossomose , Humanos , Esquistossomose/epidemiologia , Esquistossomose/tratamento farmacológico , Esquistossomose/transmissão , Animais , Malaui/epidemiologia , Enterobacteriaceae/efeitos dos fármacos , Schistosoma/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Infecções por Enterobacteriaceae/epidemiologiaRESUMO
Visceral leishmaniasis is a deadly infectious disease and is one of the world's major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE: Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans.
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Anticorpos Antiprotozoários , Antígenos de Protozoários , Leishmania donovani , Leishmaniose Visceral , Proteínas de Protozoários , Testes Sorológicos , Leishmania donovani/genética , Leishmania donovani/imunologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Animais , Humanos , Camundongos , Cães , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Testes Sorológicos/métodos , Biomarcadores/sangue , Feminino , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Camundongos Endogâmicos BALB C , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Sensibilidade e Especificidade , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologiaRESUMO
Coulomb crystals have proven to be powerful and versatile tools for the study of ion-molecule reactions under cold and controlled conditions. Reactions in Coulomb crystals are typically monitored through a combination of in situ fluorescence imaging of the laser-cooled ions and destructive time-of-flight mass spectrometry measurements of the ejected ions. However, neither of these techniques is able to provide direct structural information on the positions of nonfluorescing "dark" ions within the crystal. In this work, structural information is obtained using a phosphor screen and a microchannel plate detector in conjunction with a Timepix3 camera. The Timepix3 camera simultaneously records the spatial and temporal distribution of all ions that strike the phosphor screen detector following crystal ejection at a selected reaction time. A direct comparison can be made between the observed Timepix3 ion distributions and the distributions established from SIMION simulations of the ion trajectories through the apparatus and onto the detector. Quantitative agreement is found between the measured Timepix3 signal and the properties of Coulomb crystals assigned using fluorescence imagingâindependently confirming that the positions and numbers of nonfluorescing ions within Coulomb crystals can be accurately determined using molecular dynamics simulations. It is anticipated that the combination of high-resolution spatial and temporal data will facilitate new measurements of the ion properties within Coulomb crystals.
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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.
Assuntos
Neisseria meningitidis , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Streptococcus pneumoniae , Streptococcus pyogenes , Sequenciamento Completo do Genoma , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Humanos , Neisseria meningitidis/genética , Neisseria meningitidis/classificação , Neisseria meningitidis/isolamento & purificação , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pyogenes/genética , Streptococcus pyogenes/classificação , Filogenia , Staphylococcus aureus/genética , Genoma Bacteriano/genética , Estudo de Prova de Conceito , Bactérias/genética , Bactérias/classificação , Bactérias/isolamento & purificaçãoRESUMO
Antiviral therapeutics are highly effective countermeasures for the treatment of coronavirus disease 2019 (COVID-19). However, development of resistance to antivirals undermines their effectiveness. Combining multiple antivirals during patient treatment has the potential to overcome the evolutionary selective pressure towards antiviral resistance, as well as provide a more robust and efficacious treatment option. The current evidence for effective antiviral combinations to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication is limited. Here, we demonstrate a combination of nirmatrelvir with ombitasvir, to jointly bring about potent inhibition of SARS-CoV-2 replication. We developed an in vitro 384- well plate cytopathic effect assay for the evaluation of antiviral combinations against Calu-3 cells infected with SARS-CoV-2 and found, that a combination of ombitasvir and nirmatrelvir was synergistic; thereby decreasing the nirmatrelvir IC50 by approx. 16-fold. The increased potency of the nirmatrelvir-ombitasvir combination, over nirmatrelvir alone afforded a greater than 3 log10 reduction in viral titre, which is sufficient to fully prevent the detection of progeny SARS-CoV-2 viral particles at 48 h post infection. The mechanism of this potentiated effect was shown to be, in-part, due to joint inhibition of the 3-chymotrypsin-like protease via a positive allosteric modulation mechanism.
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COVID-19 , SARS-CoV-2 , Humanos , Anilidas , Carbamatos , Lactamas , Leucina , Nitrilas , Antivirais , RitonavirRESUMO
Aging often leads to awareness decline and psychological stress. Meditation, a method of modulating consciousness, may help individuals improve overall awareness and increase emotional resilience toward stress. This study explored the potential influence of the Awareness Training Program (ATP), a form of consciousness modulation, on age-related brain wave changes and psychological stress in middle-aged adults. Eighty-five participants with mild stress were recruited and randomly assigned to ATP (45.00 ± 8.00 yr) or control (46.67 ± 7.80 yr) groups, matched by age and gender. Ten-minute resting-state EEG data, obtained while the participants' eyes were closed, were collected using a 128-channel EEG system (EGI). A strong positive Pearson correlation was found between fast-wave (beta wave, 12-25 Hz; gamma wave, 25-40 Hz) EEG and age. However, after the 7-week ATP intervention, this correlation became insignificant in the ATP group. Furthermore, there was a significant reduction in stress levels, as measured by the Chinese version of the 10 item Perceived Stress Scale (PSS-10), in the ATP group. These results suggest that ATP may help modulate age-related effects on fast brain waves, as evidenced by the reduced correlation magnitude between age and gamma waves, and lower psychological stress. This suggests that ATP, as a form of consciousness modulation, may improve stress resilience and modulate age-related gamma wave changes.
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Companies are increasingly asking their employees to find creative solutions to their problems. However, the office environment may reduce an employee's creative potential. In this study, the role of indoor air quality parameters (PM2.5, TVOC, and CO2) in maintaining a creative environment (involving lateral thinking ability) was evaluated by Serious Brick Play (SBP), an adaptation of the LEGO Serious Play (LSP) framework. This study was conducted in a simulated office space with 92 participants over a period of 6 weeks. The SBP required participants to address a challenge by building using Lego bricks, and then describe the solution within a given timeframe. The creations and descriptions were then graded in terms of originality, fluency, and build. The results indicated that higher TVOC levels were significantly associated with lower-rated creative solutions. A 71.9% reduction in TVOC (from 1000 ppb), improves an individual's full creative potential by 11.5%. Thus, maintaining a low TVOC level will critically enhance creativity in offices.
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Poluição do Ar em Ambientes Fechados , Humanos , Pensamento , Condições de TrabalhoRESUMO
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/farmacologia , Antibacterianos/farmacologia , Plasmídeos/genética , Sequências Repetitivas Dispersas , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade MicrobianaRESUMO
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.
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Background: Brain oscillations facilitate interaction within the brain network and between the brain and heart activities, and the alpha wave, as a prominent brain oscillation, plays a major role in these coherent activities. We hypothesize that mindfully breathing can make the brain and heart activities more coherent in terms of increased connectivity between the electroencephalogram (EEG) and electrocardiogram (ECG) signals. Methods: Eleven participants (28-52 years) attended 8 weeks of Mindfulness Based Stress Reduction (MBSR) training. EEG and ECG data of two states of mindful breathing and rest, both eye-closed, were recorded before and after the training. EEGLAB was used to analyze the alpha band (8-12 Hz) power, alpha peak frequency (APF), peak power and coherence. FMRIB toolbox was used to extract the ECG data. Heart coherence (HC) and heartbeat evoked potential (HEP) were calculated for further correlation analysis. Results: After 8 weeks of MBSR training, the correlation between APF and HC increased significantly in the middle frontal region and bilateral temporal regions. The correlation between alpha coherence and heart coherence had similar changes, while alpha peak power did not reflect such changes. In contrast, spectrum analysis alone did not show difference before and after MBSR training. Conclusion: The brain works in rhythmic oscillation, and this rhythmic connection becomes more coherent with cardiac activity after 8 weeks of MBSR training. Individual APF is relatively stable and its interplay with cardiac activity may be a more sensitive index than power spectrum by monitoring the brain-heart connection. This preliminary study has important implications for the neuroscientific measurement of meditative practice.
Assuntos
Genômica , beta-Lactamases , Adulto , Humanos , Malaui/epidemiologia , beta-Lactamases/genéticaRESUMO
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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Anti-Infecciosos , Infecções por Escherichia coli , Infecções por Klebsiella , Saúde Única , Animais , Humanos , Escherichia coli , Klebsiella pneumoniae , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Estudos Longitudinais , beta-Lactamases , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/microbiologia , Estudos de CoortesRESUMO
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 , Criança , Humanos , Fluoroquinolonas/farmacologia , Tanzânia/epidemiologia , Estudos Transversais , Escherichia coli/genética , Quinolonas/farmacologiaRESUMO
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/farmacologia , Antibacterianos/farmacologia , Escherichia coli/genética , Farmacorresistência Bacteriana/genética , Plasmídeos/genética , Elementos de DNA Transponíveis , Proteínas de Escherichia coli/genética , Bactérias/genética , Testes de Sensibilidade MicrobianaRESUMO
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 , Anti-Infecciosos , Animais , Humanos , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Metagenômica , Anti-Infecciosos/farmacologia , DNARESUMO
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.