1,720,965 research outputs found
Decontamination of biofilm and VBNC zoonotic pathogens on the salad leaf phylloplane for enhanced food security and safety
No full textProduce-associated outbreaks of foodborne pathogens such as Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica are rising in prominence among outbreaks of foodborne disease. Testing for foodborne pathogens by the agricultural industry relies heavily on culture-based techniques, excluding detection of viable but nonculturable (VBNC) pathogens. Here, a detection method is used that facilitates the use of qPCR on the complex environmental matrices of soil. Targeting the tir gene of E. coli O157, detection of the pathogen in peat-based compost and sand is achieved to a sensitivity of 10 CFU/g. When applied to pristine soil, 310 copies of the gene were detected. Further analysis using PNA-FISH and cell elongation determined the presence of 205 VBNC E. coli O157 cells per gram of soil sample. Resuscitation of the pathogen was achieved through prolonged enrichment in selective media. Decontamination of fresh produce using chlorine washes was simulated using L. monocytogenes and S. enterica serovar Thompson adhered to spinach leaves, resulting in complete VBNC induction of viable cells following two minutes exposure to 50 ppm and 100 ppm chlorine respectively. The infectivity of these chlorine induced VBNC pathogens was assessed in vivo using Caenorhabditis elegans as an animal model. VBNC L. monocytogenes retained its infectivity and caused a significant lifespan reduction (p=0.0064). Together, these data provide evidence of the presence and induction of VBNC foodborne pathogens throughout the food production chain, and determines that VBNC L. monocytogenes presents a threat to food safety
Nonculturable <i>Escherichia coli</i> O157 in horticultural compost
No full textFresh produce-associated outbreaks of the foodborne pathogen Escherichia coli O157 are responsible for a number of disease cases, hospitalizations and deaths. In many cases, the source of contamination can be linked to the growing media of the food, although pathogen detection is problematic in these complex soil ecosystems. In this study, direct quantitative real-time PCR without pre-enrichment was used to detect 310 copies of the Tir gene, using a primer sequence specific to E. coli O157, in horticultural compost purchased from a commercial supplier. The pathogen could not be cultured on selective media but was visualized using peptide nucleic acid fluorescence in situ hybridization and cell elongation viability assay, confirming the viability. Enumeration of elongated E. coli O157 determined that there were 205 live cells per gram of compost. The nonculturability and confirmation of viability of the pathogen indicates its viable but nonculturable (VBNC) status. The detection of VBNC foodborne pathogens in environmental samples challenges current understanding of the nature of foodborne pathogen contamination. </p
Improved sample preparation for direct quantitative detection of Escherichia coli O157 in soil using qPCR without pre-enrichment
No full textThe prominence of fresh produce as a vehicle for foodborne pathogens such as enterohaemorrhagic Escherichia coli (EHEC) O157 is rising, where disease cases can cause hospitalisation and in some cases death. This rise emphasises the necessity for accurate and sensitive methods for detection of pathogens in soil, potential sources of contamination of fresh produce. The complexity of the soil matrix has previously proven prohibitive to pathogen detection via molecular methods without the use of a culture enrichment step, thereby excluding the detection of viable but non-culturable cells. Here, a sample preparation procedure to facilitate a direct qPCR assay is developed for the detection of E. coli O157 in soil, bypassing culture steps in favour of sample separation through pulsification release and filtration. In sand and peat-based compost, the method is sensitive to 10 CFU/g soil. When testing soils from agricultural sites, it was found that several were qPCR positive for E. coli O157 while being culture-negative, with peat-based compost possessing a concentration of 200 tir gene copies per gram. This procedure offers a rapid, quantitative assessment of the potential presence of E. coli O157 in soils which can act as a pre-screen of their suitability to grow fresh produce safely
Diagnosis and treatment of biofilm infections in children
No full textPurpose of review: biofilm-associated infections cause difficulties in the management of childhood chronic infections and other diseases, due to the invasive nature of interventions which are often necessary for definitive management. Despite their importance, there are challenges in diagnosing biofilm infections and gaps in clinicians' understanding regarding the significance of biofilms.Recent findings: many chronic infections associated with biofilms remain difficult or impossible to eradicate with conventional therapy. Surgical intervention, implant removal or long-term intermittent or suppressive antimicrobial therapy may be required. There are still significant challenges in detecting biofilms which presents a barrier in clinical practice and research. Novel therapies to disrupt biofilms are currently under investigation, which may help reduce the impact of antimicrobial resistance.Summary: biofilm-associated infection should be considered wherever there is clinical concern for an infection affecting prosthetic material, where there is a predisposing condition such as suppurative lung disease; or in the setting of chronic or relapsing infections which may be culture negative. New diagnostic methods for detecting biofilms are a research priority for both clinical diagnosis and the ability to conduct high quality clinical trials of novel antibiofilm interventions.</p
Mature Listeria monocytogenes biofilms exhibit reduced susceptibility to sanitizers - relevance to the (leafy green) fresh food supply chain
No full textSalads and herbs are important for a healthy diet but during their processing and packaging, they may be exposed to environmental contamination from foodborne pathogens. Of particular concern is Listeria monocytogenes, a Gram-positive, facultative anaerobe, found ubiquitously in the environment. L. monocytogenes can survive and grow under harsh conditions such as refrigeration temperatures, low oxygen or nutrient concentrations, which is why it is a problem in the fresh food supply chain. Infection with L. monocytogenes can result in listeriosis, potentially fatal in immunocompromised patients, pregnant women, newborn babies, and the elderly. This study aims to evaluate the efficacy of common sanitizing methods used in the fresh food supply chain, where biofilm formation has raised concerns, using appropriately developed laboratory models of Listeria biofilms. L. monocytogenes Scott A, L. monocytogenes CECT 936, and L. innocua NCTC 12210 biofilms were grown at 20 °C or 4 °C, on stainless steel coupons for 7 days, and treated with high concentrations of chlorine (up to 300 ppm) or peracetic acid (up to 500 ppm) on days 1, 3, 5, and 7. Coupons were then processed for culturable cell counts on treatment days, and imaged using episcopic differential interference microscopy, coupled with epi-fluorescence microscopy (EDIC/EF) on day 7 of growth. This determined that temperature affects biofilm growth on stainless steel, as biofilm growth reached ∼8 log
10 CFU/cm
2 at 20 °C, but was significantly lower at 4 °C (∼4 log
10 CFU/cm
2) – highlighting the importance of maintaining a cold chain. Chlorine and peracetic acid were shown to be effective at treating Listeria in the planktonic form but were not effective at treating aged biofilms at both temperatures and the high concentrations of sanitizers used. This work provides important information on sanitizing efforts in the fresh food supply chain, concerning factory temperature, processing surfaces, and the age of biofilm.</p
Viable-but-nonculturable Listeria monocytogenes and Salmonella enterica serovar Thompson induced by chlorine stress remain infectious
No full textThe microbiological safety of fresh produce is monitored almost exclusively by culture-based detection methods. However, bacterial food-borne pathogens are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses such as chlorine, which is commonly used for fresh produce decontamination. Here, complete VBNC induction of green fluorescent protein-tagged Listeria monocytogenes and Salmonella enterica serovar Thompson was achieved by exposure to 12 and 3 ppm chlorine, respectively. The pathogens were subjected to chlorine washing following incubation on spinach leaves. Culture data revealed that total viable L. monocytogenes and Salmonella Thompson populations became VBNC by 50 and 100 ppm chlorine, respectively, while enumeration by direct viable counting found that chlorine caused a <1-log reduction in viability. The pathogenicity of chlorine-induced VBNC L. monocytogenes and Salmonella Thompson was assessed by using Caenorhabditis elegans. Ingestion of VBNC pathogens by C. elegans resulted in a significant life span reduction (P = 0.0064 and P < 0.0001), and no significant difference between the life span reductions caused by the VBNC and culturable L. monocytogenes treatments was observed. L. monocytogenes was visualized beyond the nematode intestinal lumen, indicating resuscitation and cell invasion. These data emphasize the risk that VBNC food-borne pathogens could pose to public health should they continue to go undetected. IMPORTANCE Many bacteria are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses. VBNC cells cannot be detected by standard laboratory culture techniques, presenting a problem for the food industry, which uses these techniques to detect pathogen contaminants. This study found that chlorine, a sanitizer commonly used for fresh produce, induces a VBNC state in the food-borne pathogens Listeria monocytogenes and Salmonella enterica. It was also found that chlorine is ineffective at killing total populations of the pathogens. A life span reduction was observed in Caenorhabditis elegans that ingested these VBNC pathogens, with VBNC L. monocytogenes as infectious as its culturable counterpart. These data show that VBNC food-borne pathogens can both be generated and avoid detection by industrial practices while potentially retaining the ability to cause disease.</p
Multi-excitation Raman spectroscopy for label-free, strain-level characterization of bacterial pathogens in artificial sputum media
No full textThe current methods for diagnosis of acute and chronic infections are complex and skill-intensive. For complex clinical biofilm infections, it can take days from collecting and processing a patient’s sample to achieving a result. These aspects place a significant burden on healthcare providers, delay treatment, and can lead to adverse patient outcomes. We report the development and application of a novel multi-excitation Raman spectroscopy-based methodology for the label-free and non-invasive detection of microbial pathogens that can be used with unprocessed clinical samples directly and provide rapid data to inform diagnosis by a medical professional. The method relies on the differential excitation of non-resonant and resonant molecular components in bacterial cells to enhance the molecular finger-printing capability to obtain strain-level distinction in bacterial species. Here, we use this strategy to detect and characterize the respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus as typical infectious agents associated with cystic fibrosis. Planktonic specimens were analyzed both in isolation and in artificial sputum media. The resonance Raman components, excited at different wavelengths, were characterized as carotenoids and porphyrins. By combining the more informative multi-excitation Raman spectra with multivariate analysis (support vector machine) the accuracy was found to be 99.75% for both species (across all strains), including 100% accuracy for drug-sensitive and drug-resistant S. aureus. The results demonstrate that our methodology based on multi-excitation Raman spectroscopy can underpin the development of a powerful platform for the rapid and reagentless detection of clinical pathogens to support diagnosis by a medical expert, in this case relevant to cystic fibrosis. Such a platform could provide translatable diagnostic solutions in a variety of disease areas and also be utilized for the rapid detection of anti-microbial resistance
Multi-excitation raman spectroscopy complements whole genome sequencing for rapid detection of bacterial infection and resistance in WHO priority pathogens
No full textCurrent methods for diagnosing acute and complex infections mostly rely on culture-based methods and, for biofilms, fluorescence in-situ hybridization. These techniques are labor-intensive and can take 2-4 days to return a test result, especially considering an extra culturing step required for the antibiotic susceptibility testing (AST). This places a significant burden on healthcare providers, delaying treatment and leading to adverse patient outcomes. Here, we report the complementary use of our newly developed multi-excitation Raman spectroscopy (ME-RS) method with whole-genome sequencing (WGS). Four WHO priority pathogens are AST phenotyped and their antimicrobial resistance (AMR) profile determined by WGS. On application of ME-RS method we find high correlation with the WGS characterization. Highly accurate classification based on the species (98.93%), wild-type/non-wild type (99.45%), and presence or absence of thick peptidoglycan layers in cell walls (100%), as well as at the individual strain level (99.29%). These results clearly demonstrate the potential of ME-RS as a rapid and first-stage tool for species, resistance and strain-level classification which can be followed up by WGS for confirmation. Such a workflow can facilitate efficient antimicrobial stewardship to handle and prevent the spread of AMR
Enhanced Raman techniques for infection diagnostics
No full textIn this paper we describe our recent work in multi-excitation surface enhanced Raman spectroscopy (MX-SERS), and its application for robust strain-level bacteria identification. The development of MX-SERS follows directly from our previous work in rapid bacterial identification using multi-excitation Raman spectroscopy (MX-Raman), which enabled highly accurate (up to 99.75%) strain-level distinction of bacteria, including antibiotic resistant strains of bacteria and from within complex media. In this work we use the strong wavelength dependence of both the Raman scattering cross-section and the surface plasmon to demonstrate a novel capability in bacteria identification. Compared to MX-Raman, MX-SERS has up to 8x faster data acquisition speed as well as up to 4000x lower laser power incident on the sample. Furthermore, we fabricate SERS-active substrates with a simple and low-cost fabrication method that can be adapted to fit a chosen wavelength regime. This combination of strain-level sensitivity and high-speed detection, combined with a low-cost SERS substrate, has strong potential applications in clinical diagnostics, and could be integrated within a real-world pathogen detection workflow. This presents new capabilities in label-free bacterial detection including novel culture-free investigation capabilities, and an improved methodology for sample handling with minimal preparation and processing.</p
- …
