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Pathogenic microbes invade the human body and trigger a host immune response to defend against the infection. In response, host-adapted pathogens employ numerous virulence strategies to overcome host defense mechanisms. As a result, the interaction between the host and pathogen is a dynamic process that shapes the evolution of the host's immune response. Among the immune responses against intracellular bacteria, pyroptosis, a lytic form of cell death, is a crucial mechanism that eliminates replicative niches for intracellular pathogens and modulates the immune system by releasing danger signals. This review focuses on the role of pyroptosis in combating intracellular bacterial infection. We examine the cell type specific roles of pyroptosis in neutrophils and intestinal epithelial cells. We discuss the regulatory mechanisms of pyroptosis, including its modulation by autophagy and interferon-inducible GTPases. Furthermore, we highlight that while host-adapted pathogens can often subvert pyroptosis, environmental microbes are effectively eliminated by pyroptosis.
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Infecções Bacterianas , Piroptose , Humanos , Morte Celular , Neutrófilos , BactériasRESUMO
Biofilm formation is likely to contribute greatly to antibiotic resistance in bacteria and therefore the efficient removal of bacterial biofilms needs addressing urgently. Here, we reported that the supplement of non-inhibitory concentration of N-acetyl-L-cysteine (NAC), a common reactive oxygen species (ROS) scavenger, can significantly reduce the biomass of mature Pseudomonas aeruginosa biofilms (corroborated by crystal violet assay and laser scanning confocal microscopy). 1 mM NAC increased the cheater (ΔlasR mutant) frequency to 89.4 ± 1.5% in the evolved PAO1 after the 15-day treatment. Scavenging of ROS by NAC induced the collapse of P. aeruginosa biofilms, but it did not alter quorum sensing-regulated genes expression (e.g., hcnC and cioAB) and hydrogen cyanide production. The replenishment of public good protease contributed to the recovery of biofilm biomass, indicating the role of disrupting policing in biofilm inhibition. Furthermore, 7 typical ROS scavengers (e.g., superoxide dismutase, catalase and peroxidase, etc.) also effectively inhibited mature P. aeruginosa biofilms. This study demonstrates that scavenging of ROS can promote the selective control of P. aeruginosa biofilms through policing disruption as a targeted biofilm control strategy in complex water environments.
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Antibacterianos , Pseudomonas aeruginosa , Espécies Reativas de Oxigênio/metabolismo , Antibacterianos/farmacologia , Pseudomonas aeruginosa/genética , Biofilmes , Resistência Microbiana a Medicamentos , Acetilcisteína/farmacologiaRESUMO
Disease outbreaks and pathogen introductions can have significant effects on host populations, and the ability of pathogens to persist in the environment can exacerbate disease impacts by fueling sustained transmission, seasonal epidemics, and repeated spillover events. While theory suggests that the presence of an environmental reservoir increases the risk of host declines and threat of extinction, the influence of reservoir dynamics on transmission and population impacts remains poorly described. Here we show that the extent of the environmental reservoir explains broad patterns of host infection and the severity of disease impacts of a virulent pathogen. We examined reservoir and host infection dynamics and the resulting impacts of Pseudogymnoascus destructans, the fungal pathogen that causes white-nose syndrome, in 39 species of bats at 101 sites across the globe. Lower levels of pathogen in the environment consistently corresponded to delayed infection of hosts, fewer and less severe infections, and reduced population impacts. In contrast, an extensive and persistent environmental reservoir led to early and widespread infections and severe population declines. These results suggest that continental differences in the persistence or decay of P. destructans in the environment altered infection patterns in bats and influenced whether host populations were stable or experienced severe declines from this disease. Quantifying the impact of the environmental reservoir on disease dynamics can provide specific targets for reducing pathogen levels in the environment to prevent or control future epidemics.
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Quirópteros/microbiologia , Reservatórios de Doenças/microbiologia , Micoses/epidemiologia , Animais , Ascomicetos/patogenicidade , Epidemias , Hibernação , Micoses/microbiologia , Nariz/microbiologia , Doenças Nasais/epidemiologia , Doenças Nasais/microbiologia , Dinâmica Populacional , Estações do AnoRESUMO
During its life cycle, the environmental pathogen Legionella pneumophila alternates between a replicative and transmissive cell type when cultured in broth, macrophages, or amoebae. Within a protozoan host, L. pneumophila further differentiates into the hardy cell type known as the mature infectious form (MIF). The second messenger cyclic di-GMP coordinates lifestyle changes in many bacterial species, but its role in the L. pneumophila life cycle is less understood. Using an in vitro broth culture model that approximates the intracellular transition from the replicative to the transmissive form, here we investigate the contribution to L. pneumophila differentiation of a two-component system (TCS) that regulates cyclic di-GMP metabolism. The TCS is encoded by lpg0278-lpg0277 and is cotranscribed with lpg0279, which encodes a protein upregulated in MIF cells. The promoter for this operon is RpoS dependent and induced in nutrient-limiting conditions that do not support replication, as demonstrated using a gfp reporter and quantitative PCR (qPCR). The response regulator of the TCS (Lpg0277) is a bifunctional enzyme that both synthesizes and degrades cyclic di-GMP. Using a panel of site-directed point mutants, we show that cyclic di-GMP synthesis mediated by a conserved GGDEF domain promotes growth arrest of replicative L. pneumophila, accumulation of pigment and poly-3-hydroxybutyrate storage granules, and viability in nutrient-limiting conditions. Genetic epistasis tests predict that the MIF protein Lpg0279 acts as a negative regulator of the TCS. Thus, L. pneumophila is equipped with a regulatory network in which cyclic di-GMP stimulates the switch from a replicative to a resilient state equipped to survive in low-nutrient environments.IMPORTANCE Although an intracellular pathogen, L. pneumophila has developed mechanisms to ensure long-term survival in low-nutrient aqueous conditions. Eradication of L. pneumophila from contaminated water supplies has proven challenging, as outbreaks have been traced to previously remediated systems. Understanding the genetic determinants that support L. pneumophila persistence in low-nutrient environments can inform design and assessment of remediation strategies. Here we characterize a genetic locus that encodes a two-component signaling system (lpg0278-lpg0277) and a putative regulator protein (lpg0279) that modulates the production of the messenger molecule cyclic di-GMP. We show that this locus promotes both L. pneumophila cell differentiation and survival in nutrient-limiting conditions, thus advancing the understanding of the mechanisms that contribute to L. pneumophila environmental resilience.
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Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Regulação Bacteriana da Expressão Gênica , Legionella pneumophila/fisiologia , Viabilidade Microbiana , Aminoácidos/metabolismo , Meios de Cultura , GMP Cíclico/genética , GMP Cíclico/metabolismo , Hidroxibutiratos/metabolismo , Legionella pneumophila/genética , Poliésteres/metabolismo , Transdução de SinaisRESUMO
Wild banded mongooses ( Mungos mungo) in northeastern Botswana and northwest Zimbabwe are infected with a novel Mycobacterium tuberculosis complex (MTC) pathogen, Mycobacterium mungi. We evaluated gross and histologic lesions in 62 infected mongooses (1999-2017). Many tissues contained multifocal irregular, lymphohistiocytic to granulomatous infiltrates and/or multifocal or coalescing noncaseating to caseating granulomas with variable numbers of intralesional acid-fast bacilli. Over one-third of nasal turbinates examined had submucosal lymphohistiocytic to granulomatous infiltrates, erosion and ulceration of the nasal mucosa, bony remodeling, and nasal distortion. Similar inflammatory cell infiltrates expanded the dermis of the nasal planum with frequent ulceration. However, even in cases with intact epidermis, acid-fast bacilli were present in variable numbers among dermal infiltrates and on the epidermal surface among desquamated cells and debris, most commonly in small crevices or folds. In general, tissue involvement varied among cases but was highest in lymph nodes (50/54, 93%), liver (39/53, 74%), spleen (37/51, 73%), and anal glands/sacs (6/8, 75%). Pulmonary lesions were present in 67% of sampled mongooses (35/52) but only in advanced disseminated disease. The pathological presentation of M. mungi in the banded mongoose is consistent with pathogen shedding occurring through scent-marking behaviors (urine and anal gland secretions) with new infections arising from contact with these contaminated olfactory secretions and percutaneous movement of the pathogen through breaks in the skin, nasal planum, and/or skin of the snout. Given the character and distribution of lesions and the presence of intracellular acid-fast bacilli, we hypothesize that pathogen spread occurs within the body through a hematogenous and/or lymphatic route. Features of prototypical granulomas such as multinucleated giant cells and peripheral fibrosis were rarely present in affected mongooses. Acid-fast bacilli were consistently found intracellularly, even in regions of necrosis. The mongoose genome has a unique deletion (RD1mon) that includes part of the encoding region for PPE68 (Rv3873), a gene co-operonic with PE35. These proteins can influence the host's cellular immune response to mycobacterial infections, and it remains uncertain how this deletion might contribute to observed patterns of pathology. M. mungi infection in banded mongooses is characterized by both a unique transmission and exposure route, as well as accompanying pathological features, providing an opportunity to increase our understanding of MTC pathogenesis across host-pathogen systems.
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Herpestidae/microbiologia , Infecções por Mycobacterium/veterinária , Mycobacterium , Sacos Anais/patologia , Animais , Feminino , Fígado/patologia , Pulmão/patologia , Linfonodos/patologia , Masculino , Infecções por Mycobacterium/microbiologia , Infecções por Mycobacterium/patologia , Pele/patologia , Baço/patologiaRESUMO
After the deaths of 2 preterm neonates with Bacillus cereus systemic infection in the same intensive care unit, we investigated the pathogenic potential of this bacterium. Genetic and virulence analysis indicated the neonates were infected with 2 different strains with a virulence potential similar to environmental strains, indicating likely patient immune response failure.
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Bacillus cereus/isolamento & purificação , Infecções por Bactérias Gram-Positivas/diagnóstico , Infecções por Bactérias Gram-Positivas/microbiologia , Antibacterianos/uso terapêutico , Bacillus cereus/genética , Bacillus cereus/patogenicidade , Infecção Hospitalar , Quimioterapia Combinada , Evolução Fatal , Feminino , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Humanos , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Gravidez , Ultrassonografia Pré-Natal , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Environmental pathogen surveillance is a promising disease surveillance modality that has been widely adopted for SARS-CoV-2 monitoring. The highly variable nature of environmental pathogen data is a challenge for integrating these data into public health response. One source of this variability is heterogeneous infection both within an individual over the course of infection as well as between individuals in their pathogen shedding over time. We present a mechanistic modeling and estimation framework for connecting environmental pathogen data to the number of infected individuals. Infected individuals are modeled as shedding pathogen into the environment via a Poisson process whose rate parameter λt varies over the course of their infection. These shedding curves λt are themselves random, allowing for variation between individuals. We show that this results in a Poisson process for environmental pathogen levels with rate parameter a function of the number of infected individuals, total shedding over the course of infection, and pathogen removal from the environment. Theoretical results include determination of identifiable parameters for the model from environmental pathogen data and simple, explicit formulas for the likelihood for particular choices of individual shedding curves. We give a two step Bayesian inference framework, where the first step corresponds to calibration from data where the number of infected individuals is known, followed by an estimation step from environmental surveillance data when the number of infected individuals is unknown. We apply this modeling and estimation framework to synthetic data, as well as to an empirical case study of SARS-CoV-2 in environmental dust collected from isolation rooms housing university students. Both the synthetic data and empirical case study indicate high inter-individual variation in shedding, leading to wide credible intervals for the number of infected individuals. We examine how uncertainty in estimates of the number of infected individuals from environmental pathogen levels scales with the true number of infected individuals and model misspecification. While credible intervals for the number of infected individuals are wide, our results suggest that distinguishing between no infection and small-to-moderate levels of infection (≈10 infected individuals) may be possible, and that it is broadly possible to differentiate between moderate (≈40) and high (≈200) numbers of infected individuals.
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Listeriosis is a disease caused by L. monocytogenes, a relevant microorganism as a causative agent of foodborne diseases - FBD. This study aimed to evaluate the distribution of Listeria spp., and L. monocytogenes in different production areas in two small plants (A and B) and two micro-food processing plants (C and D) producing meat derivatives, located in different cities of Colombia. The methodology implemented was i. The analysis of sampling points is based on a harmonised tool. ii. Four samplings in each production plant between 2019 and 2020. iii. Isolation and identification of microorganisms through conventional microbiology, a semi-automated system, molecular serotyping and clonal characterisation by ERIC-PCR. L. monocytogenes frequency in the production plants belonging to the study ranged between 5.9 and 28.6 %; for Listeria spp., plants A and D had isolated, plant A had the highest proportion, while for L. monocytogenes geno-serotypes found were: 1/2a, 1/2c, 4a-4c, 4b, 4d - 4e, with geno-serotype 4b as the most frequent. Furthermore, possible persistent isolates were detected in plant C as the feasible sources of contamination, based on failures in flow management, raw material contaminated with L. monocytogenes, lack of standardised cooking processes and transfer of the microorganism through equipment and surfaces. Finally, in three of the four production plants assayed, L. monocytogenes or Listeria spp. were present in the packaging area in some of the samples taken during the study, which calls for increased and frequent monitoring, as well as constant technical support for the control of L. monocytogenes in micro and small-scale production plants.
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Enterococcus has been considered one of the most important nosocomial pathogens for human infections, and the hospital environment is an important reservoir for vancomycin-resistant enterococci (VRE) that leads to antimicrobial therapeutic failure. However, infant foods and their production environments could pose risks for the immature population, while this question remains unaddressed. This study conducted an extensive and thorough Enterococcus isolation, VRE risk assessment of the Chinese infant food production chains and additional online-marketing infant foods, including powdered infant formula (PIF) and infant complementary food (ICF). To investigate the prevalence of Enterococcus along infant food chains and commodities, a total of 482 strains of Enterococcus, including E. faecium (n = 363), E. faecalis (n = 84), E. casseliflavus (n = 13), E. mundtii (n = 12), E. gallinarum (n = 4), E. hirae (n = 4), and E. durans (n = 2) were recovered from 459 samples collected from infant food production chains (71/254) and food commodities (67/205). A decreasing trend for Enterococcus detection rate was found in the PIF production chain (PIF-PC), particularly during the preparation of the PIF base powder (From 100 % in raw milk to 8.70 % in end products), while an increasing trend was observed in the ICF production chain (ICF-PC) mainly during the initial processing of farm crops and the further processing of the product (20 % at farm crops increasing to 76.92 % at end products). The result indicated that the PIF-PC process effectively reduced Enterococcus contamination, while the ICF-PC showed the opposite trend. Importantly, eleven VRE isolates were recovered from the infant food production chain, including seven E. casseliflavus isolates carrying vanC2/C3 and four E. gallinarum isolates carrying vanC1. Ten VRE isolates were from food production environments. Collectively, our study demonstrated that infant food production environments represent potential reservoirs for VRE non-nosocomial infections in vulnerable populations.
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Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Enterococos Resistentes à Vancomicina , Humanos , Vancomicina , Resistência a Vancomicina , Antibacterianos/farmacologia , Fórmulas Infantis , Infecções por Bactérias Gram-Positivas/epidemiologia , Testes de Sensibilidade MicrobianaRESUMO
Mycobacterium ulcerans is the causative agent of Buruli ulcer - a necrotizing skin infection. As an environmental pathogen, it has developed stress response mechanisms for survival. Similar to endospore formation in M. marinum, it is likely that M. ulcerans employs sporulation mechanisms for its survival and transmission. In this review, we modeled possible transmission routes and patterns of M. ulcerans from the environment to its host. We provided insights into the evolution of M. ulcerans and its genomic profiles. We discuss reservoirs of M. ulcerans as an environmental pathogen and its environmental survival. We comprehensively discuss sporulation as a possible stress response mechanism and modelled endospore formation in M. ulcerans. At last, we highlighted sporulation associated markers, which upon expression trigger endospore formation.
Buruli ulcer is an infectious disease characterized by extensive sores on the skin and soft body tissues. The disease is caused by a bacterium called Mycobacterium ulcerans and is mainly found in tropical countries. Over the years, several attempts to understand the means by which humans get into contact with this bug as well as how it thrives in its host remain futile. In this review, we describe a possible survival strategy, known as sporulation, that is adopted by the pathogen for dispersal and survival.
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Environmental pathogen reservoirs exist for many globally important diseases and can fuel epidemics, influence pathogen evolution, and increase the threat of host extinction. Species composition can be an important factor that shapes reservoir dynamics and ultimately determines the outcome of a disease outbreak. However, disease-induced mortality can change species communities, indicating that species responsible for environmental reservoir maintenance may change over time. Here we examine the reservoir dynamics of Pseudogymnoascus destructans, the fungal pathogen that causes white-nose syndrome in bats. We quantified changes in pathogen shedding, infection prevalence and intensity, host abundance, and the subsequent propagule pressure imposed by each species over time. We find that highly shedding species are important during pathogen invasion, but contribute less over time to environmental contamination as they also suffer the greatest declines. Less infected species remain more abundant, resulting in equivalent or higher propagule pressure. More broadly, we demonstrate that high infection intensity and subsequent mortality during disease progression can reduce the contributions of high-shedding species to long-term pathogen maintenance.
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Pathogens in our environment can act as agents capable of inflicting severe human diseases. Among them, the SARS-CoV-2 virus has recently plagued the globe and paralyzed the functioning of ordinary human life. The virus enters the cell through the angiotensin-converting enzyme-2 (ACE-2) receptor, an integral part of the renin-angiotensin system (RAAS). Reports on hypertension and its relation to the modulation of the RAAS are generating interest in the scientific community. This short review focuses on the SARS-CoV-2 infection's direct and indirect effects on our body through modulation of the RAAS axis. A patient having severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection, which causes COVID-19 relates to hypertension as a pre-existing disease or develops it in a post-COVID scenario. Several studies on how SARS-CoV-2 modulates the RAAS axis indicate that it alters our body's physiological balance. This review seeks to establish a hypothesis on the mechanical dependency of SARS-CoV-2 and RAAS modulation in the human body. This study intends to impart ideas on drug development and designing by targeting the modulation of the RAAS axis to inactivate the pathogenicity of the SARS-CoV-2 virus. A systematic hypothesis can severely attenuate the pathogenicity of the dreadful viruses of the future.
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COVID-19 , Hipertensão , Aldosterona/farmacologia , Angiotensinas/farmacologia , Humanos , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/farmacologia , Renina/farmacologia , Sistema Renina-Angiotensina/fisiologia , SARS-CoV-2RESUMO
Mastitis is the most prevalent and economically important disease caused by different etiological agents, which leads to increased somatic cell count (SCC) and low milk quality. Treating mastitis cases with antimicrobials is essential to reduce SCC and improve milk quality. Non-prudent use of antimicrobials in dairy farms increased the development of antimicrobial resistant bacteria. This study's objectives were (1) to isolate and identify etiological agents of mastitis and (2) to determine antimicrobial resistance profiles of bacterial isolates. A total of 174 quarter milk samples from 151 cows with high SCC and clinical mastitis from 34 dairy farms in Tennessee, Kentucky, and Mississippi were collected. Bacterial causative agents were determined by bacteriological and biochemical tests. The antimicrobial resistance of bacterial isolates against 10 commonly used antimicrobials was tested. A total of 193 bacteria consisting of six bacterial species, which include Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli, Klebsiella oxytoca and Klebsiella pneumoniae were isolated. Staphylococcus aureus was the predominant isolate followed by Strep. spp., E. coli, and Klebsiella spp. Results of this study showed that Gram-negatives (E. coli and Klebsiella spp.) were more resistant than Gram-positives (Staph. aureus and Streptococcus spp.). Continuous antimicrobial resistance testing and identification of reservoirs of resistance traits in dairy farms are essential to implement proper mitigation measures.
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Since March 2020, Spain (along with many other countries) has been severely affected by the ongoing coronavirus disease 19 (COVID-19) pandemic caused by the rapid spread of a new virus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2). As part of global efforts to improve disease surveillance, we investigated how readily SARS-CoV-2 RNA could be detected in environmental samples collected from an isolated rural community in Spain with a high COVID-19 prevalence (6% of the population of 883 inhabitants). The first diagnosis of COVID-19-compatible symptoms in the village was recorded on 3 March 2020, and the last known active case resolved on 5 June 2020. By 15 May, two months after strict movement constraints were imposed ('lockdown'), and the cumulative number of symptomatic cases had increased to 53. Of those cases, 22 (41%) had been tested and confirmed by RT-PCR. On 13 May and 5 June, samples were collected from high-use surfaces and clothes in the homes of 13 confirmed cases, from surfaces in nine public service sites (e.g. supermarket and petrol station) and from the wastewater of the village sewage system. SARS-CoV-2 RNA was detected in 7 of 57 (12%) samples, including three households and three public sites. While there is not yet sufficient evidence to recommend environmental surveillance as a standard approach for COVID-19 epidemiology, environmental surveillance research may contribute to advance knowledge about COVID-19 by further elucidating virus shedding dynamics and environmental contamination, including the potential identification of animal reservoirs.
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COVID-19/epidemiologia , Microbiologia Ambiental , Monitoramento Ambiental , RNA Viral/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Animais , COVID-19/virologia , Controle de Doenças Transmissíveis , Humanos , Prevalência , SARS-CoV-2/genética , Espanha/epidemiologia , Eliminação de Partículas ViraisRESUMO
The limit of detection (LOD) for qPCR-based analyses is not consistently defined or determined in studies on waterborne pathogens. Moreover, the LODs reported often reflect the qPCR assay alone rather than the entire sample process. Our objective was to develop an approach to determine the 95% LOD (lowest concentration at which 95% of positive samples are detected) for the entire process of waterborne pathogen detection. We began by spiking the lowest concentration that was consistently positive at the qPCR step (based on its standard curve) into each procedural step working backwards (i.e., extraction, secondary concentration, primary concentration), which established a concentration that was detectable following losses of the pathogen from processing. Using the fraction of positive replicates (n = 10) at this concentration, we selected and analyzed a second, and then third, concentration. If the fraction of positive replicates equaled 1 or 0 for two concentrations, we selected another. We calculated the LOD using probit analysis. To demonstrate our approach we determined the 95% LOD for Salmonella enterica serovar Typhimurium, adenovirus 41, and vaccine-derived poliovirus Sabin 3, which were 11, 12, and 6 genomic copies (gc) per reaction (rxn), respectively (equivalent to 1.3, 1.5, and 4.0 gc L(-1) assuming the 1500 L tap-water sample volume prescribed in EPA Method 1615). This approach limited the number of analyses required and was amenable to testing multiple genetic targets simultaneously (i.e., spiking a single sample with multiple microorganisms). An LOD determined this way can facilitate study design, guide the number of required technical replicates, aid method evaluation, and inform data interpretation.
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Limite de Detecção , Microbiologia da Água , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , ÁguaRESUMO
This paper focuses on potential hazards and risks to forensic anthropologists while working in the field and laboratory in North America. Much has changed since Galloway and Snodgrass published their seminal article addressing these issues. The increased number of forensic practitioners combined with new information about potential hazards calls for an updated review of these pathogens and chemicals. Discussion of pathogen hazards (Brucella, Borrelia burgdorferi, Yersinia pestis, Clostridium tetani and West Nile virus) includes important history, exposure routes, environmental survivability, early symptoms, treatments with corresponding morbidity and mortality rates, and decontamination measures. Additionally, data pertaining to the use of formaldehyde in the laboratory environment have resulted in updated safety regulations, and these are highlighted. These data should inform field and laboratory protocols. The hazards of working directly with human remains are discussed in a companion article, "An Update on the Hazards and Risks of Forensic Anthropology, Part I: Human Remains."
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Antropologia Forense , Exposição Ocupacional , Substâncias Perigosas , Humanos , Laboratórios , América do Norte , RiscoRESUMO
We present a reliable PCR-based method to avoid the biases related to identification based on the conventional phenotypes currently used in the identification of Pseudomonas syringae sensu lato, a ubiquitous environmental bacterium including plant pathogens. We identified a DNA target suitable for this purpose by applying a comparative genomic pipeline to Pseudomonas genomes. We designed primers and developed PCR conditions that led to a clean and strong PCR product from 97% of the 185 strains of P. syringae strains tested and gave a clear negative result for the 31 non-P. syringae strains tested. The sensitivity of standard PCR was determined with pure strains to be 10(6) bacteria mL(-1) or 0.4 ng of DNA µL(-1). Sensitivity could be improved with the touchdown method. The new PCR-assisted isolation of P. syringae was efficient when deployed on an environmental sample of river water as compared to the isolation based on phenotypes. This innovation eliminates the need for extensive expertise in isolating P. syringae colonies, was simpler, faster and very reliable. It will facilitate discovery of more diversity of P. syringae and research on emergence, dispersion and evolution to understand the varied functions of this environmental bacterium.