Low Concentrations Of Bacteria Research Articles

Preparation and Application of pH‐Sensitive Protein Nanofibre Membrane Loaded With Alizarin and Curcumin for Meat Preservation

ABSTRACTA pH‐sensitive protein fibre membrane was prepared using alizarin (ALI), curcumin (CUR), gelatin, zein and cinnamaldehyde as raw materials, by electrospinning technology. The membrane was developed to detect food deterioration and extend shelf life. It was observed that the addition of CUR significantly enhanced the antioxidant and barrier properties of the membrane. The fibre membrane was responsive to pH with minimal colour alteration in response to humidity (from 20% to 80% relative humidity) and demonstrated excellent reusability along with a low detection limit for ammonia gas. Moreover, it was capable of monitoring low concentrations of bacteria in the headspace. To assess the efficacy of the membrane, a freshness monitoring and preservation study was carried out on pork stored at 4°C for 5 days. The fibre membrane not only prolonged the shelf life of meat but also allowed real‐time monitoring of its freshness. The results highlight the potential application of the prepared nanofibres in the simultaneous monitoring and preservation of meat freshness.

Cascade Structured Plasmonic Liquid Crystal Biosensor for the Rapid Detection of Harmful Bacteria Dispersed in Potable Water

AbstractPathogenic microorganisms contaminating potable water are a serious water quality concern because they have severe consequences for human and environmental health. Managing water contamination requires the availability of fast and highly sensitive point‐of‐use detection systems responsive to a wide concentration range. In the present work, this goal is achieved by realizing a cascade‐structured biosensor that exploits innovative stimuli‐responsive materials such as gold nanorods (AuNRs) and photosensitive nematic liquid crystals (NLCs). The cascade structure is fabricated by interfacing a glass substrate in a back‐to‐front arrangement, hosting an array of bioactivated AuNRs and an NLC cell. The AuNRs array integrates microfluidic channels, allowing direct water sampling and the analysis of reduced water volumes with high sensitivity. The biosensor combines in the same device two independent optical transducers: a bioactive AuNRs array (plasmonic biosensor), sensitive to refractive index alterations, and an NLC cell that detects the presence of pathogens by responding to light intensity variations. The plasmonic biosensor performs exceptionally well for very low concentrations of bacteria. In contrast, the NLC biosensor works for high‐concentration bacteria, thus providing a cascade‐like detection system able to detect bacteria in a wide concentration range from 10 to 109 CFU mL−1.

Enhancing pathogen identification in cheese with high background microflora using an artificial neural network-enabled paper chromogenic array sensor approach

Biohazards, which may occur at all supply chain stages, pose significant threats to food safety and public health. Addressing these concerns and enhancing food safety necessitates a nondestructive pathogen surveillance approach capable of continuously and simultaneously detecting multiple pathogens. Detecting and differentiating low concentrations of pathogenic bacteria amid high background microflora levels in foods is challenging, requiring technology with high sensitivity and robust discriminatory capability. This study introduces an artificial neural network-driven paper chromogenic array sensor (ANN-PCA) technique developed for the nondestructive, continuous, and simultaneous detection of Salmonella Enteritidis (SE) and Escherichia coli O157:H7 (Ec) from a high background microflora in shredded cheddar cheese. This method enables accurate detection of SE and Ec in monoculture and cocktail culture while distinguishing them from a high level of background microflora (∼7.5 log CFU/g), with accuracies ranging from 72 ± 11% to 92 ± 3%. In addition, SE and Ec were successfully identified at concentrations as low as 1 log CFU/g within one day, with an accuracy of 72 ± 11%. This approach exhibits promising potential for integration into a digitalized, smart, and resilient nondestructive surveillance system for real-time pathogen detection in foods throughout the supply chain without enrichment, incubation, or other sample preparation steps.

Improving the microbial and physicochemical shelf life of yufka paste using Lactobacillus plantarum and calcium propionate.

Considering the importance of reducing the use of chemical preservatives in food and the increasing attention of consumers to consume food products with minimal additives, the main goal of this research was to study the effect of use of chemical (calcium propionate) and biological (Lactobacillus plantarum) preservatives on increasing shelf life of yufka paste considering its physicochemical and microbial characteristics. For this purpose, three samples of yufka paste were prepared by adding concentrations of 103, 105, and 107 cfu/mL of L. plantarum individually and three samples of paste were prepared by adding the same amount of bacteria in combination with 0.3% calcium propionate and these samples were compared with the control sample (without preservative) and the sample only containing 0.3% calcium propionate. The obtained results showed that different treatments and time had a significant effect on physicochemical properties including pH, moisture, and protein of yufka paste (p < .05). The results of the survival of L. plantarum showed that with increasing time, the survival rate of bacteria increased (p < .05). The pH of the samples showed that the L. plantarum has a significant effect on controlling the chemical quality of yufka during storage. The count of mold and yeast in the combined use of L. plantarum and 0.3% propionate was lower than the single use of propionate chemical preservative, which indicated the very good effect of the green preservative in controlling the moldy spoilage of yufka. Low concentrations of bacteria showed better antimold results than treatments containing bacteria and propionate in a combined form, propionate or control treatment.

UtPCR: A Strategy for the Highly Specific and Absolutely Quantitative Detection of Single Molecules within Only Minutes.

Bloodstream infection is a major health problem worldwide, with extremely high mortality. Detecting infection in the early stage is challenging due to the extremely low concentration of bacteria in the blood. Digital PCR provides unparalleled sensitivity and can achieve absolute quantification, but it is time-consuming. Moreover, the presence of unavoidable background signals in negative controls poses a significant challenge for single-molecule detection. Here, we propose a novel strategy called "Ultrafast flexible thin tube-based droplet digital PCR (utPCR)" that can shorten the digital PCR process from 2 h to only 5 min, with primer annealing/extension time reduced from minutes to only 5 s. Importantly, the ultrafast PCR eliminates nonspecific amplification and thus enables single-molecule detection. The utPCR enabled the sensitive detection and digital quantification of E. coli O157 in the high background of a 106-fold excess of E. coli K12 cells. Moreover, this method also displayed the potential to detect rare pathogens in blood samples, and the limit of detection (LOD) could be as low as 10 CFU per mL of blood without false positive results. Considered ultrafast (<5 min) and highly sensitive (single-molecule detection), the utPCR holds excellent prospects in the next generation of molecular diagnosis.

Detailed Mechanisms Underlying Neutrophil Bactericidal Activity against Streptococcus pneumoniae.

Neutrophils are an essential cellular component of innate immunity and control bacterial infections through a combination of intracellular and extracellular killing methods. Although the importance of neutrophils has been established, the exact methods used to handle particular bacterial challenges and the efficiency of bacterial killing remain not well understood. In this study, we addressed how neutrophils eliminate Streptococcus pneumoniae (Spn), a leading cause of community acquired and post-influenza bacterial pneumonia. We analyzed killing methods with variable bacterial:neutrophil concentrations and following priming with PAM3CSK4 (P3CSK), an agonist for Toll-like-receptor 2 (TLR2). Our results show that murine neutrophils display surprisingly weak bactericidal activity against Spn, employing a predominantly extracellular mode of killing at lower concentrations of bacteria, whereas challenges with higher bacterial numbers induce both extracellular and intracellular elimination modes but require TLR2 activation. TLR2 activation increased reactive oxygen species (ROS) and neutrophil extracellular trap (NET) formation in response to Spn. Despite this, supernatants from P3CSK-stimulated neutrophils failed to independently alter bacterial replication. Our study reveals that unstimulated neutrophils are capable of eliminating bacteria only at lower concentrations via extracellular killing methods, whereas TLR2 activation primes neutrophil-mediated killing using both intracellular and extracellular methods under higher bacterial burdens.

Ultrasensitive Detection of Multidrug-Resistant Bacteria Based on Boric Acid-Functionalized Fluorescent MOF@COF.

The widespread use of antibiotics has made multidrug-resistant bacteria (MDRB) one of the greatest threats toward global health. Current conventional microbial detection methods are usually time-consuming, labor-intensive, expensive, and unable to detect low concentrations of bacteria, which cause great difficulties in clinical diagnosis and treatment. Herein, we constructed a versatile biosensing platform on the basis of boric acid-functionalized porous framework composites (MOF@COF-BA), which were able to realize highly efficient and sensitive label-free MDRB detection via fluorescence. In this design, MDRB were captured using aptamer-coated nanoparticles and the fluorescent probe MOF@COF-BA was tightly anchored onto the surface of MDRB due to interactions between boric acid groups and glycolipids on bacteria cells. Benefitting from the remarkable fluorescence performance of MOF@COF-BA, rapid and specific detection of MDRB, such as methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii (AB), was realized with a detection range of 20-108 CFU/mL (for both) and limits of detection of 7 CFU/mL (MRSA) and 5 CFU/mL (AB). The feasibility of using the developed platform to selectively detect MRSA and AB from complex urine, human serum, and cerebrospinal fluid samples was also demonstrated. This work provides a promising strategy for accurate MDRB diagnosis, avoiding serious infection using rational antibiotic therapy.

Aerosol partitioning potential of bacteria presenting antimicrobial resistance from different stages of a small decentralized septic treatment system

The release of airborne pathogens with antimicrobial resistance is a growing global public health threat. The potential spread of antimicrobial resistant bacteria and antimicrobial resistance genes (ARB/ARGs) from wastewater effluents through aerosolization is not fully understood. Septic tanks are on-site wastewater treatment systems (OWTS) used in about 25% of households in the United States (US). The system in this study consists of four tanks connected by two different treatment types, with wastewater moving from the most contaminated, Tank 1, to the most purified holding, Tank 4. Aerosol samples at each tank were collected in the summer and winter with open and closed lids to observe their effect on bioaerosol emissions. The water inside each tank was also sampled to compare the bacteria concentration and composition. Results from microbial and molecular analysis indicate lower concentrations of bacteria in the aerosols compared to the water samples. The antibiotic testing indicates that aerosolized bacteria from the winter were more resistant to the antibiotics used compared to the aerosolized bacteria from the summer. The occurrence of aerosolized bacteria likely depends on operational conditions and discharge methods, on changes in humidity, temperature, and air velocity, while the effluent resistance can be influenced by quorum sensing. Further assessment of the water and bioaerosol microbiome should include the genomic analysis of antimicrobial resistance of bacterial isolates from both sources and thus provide an important link regarding human exposure to ARBs/ARGs near wastewater treatment systems.Copyright © 2023 American Association for Aerosol Research

Efficient filter-in-centrifuge separation of low-concentration bacteria from blood.

Separating bacteria from infected blood is an important step in preparing samples for downstream bacteria detection and analysis. However, the extremely low bacteria concentration and extremely high blood cell count make efficient separation challenging. In this study, we introduce a method for separating bacteria from blood in a single centrifugation step, which involves sedimentation velocity-based differentiation followed by size-based cross-flow filtration over an inclined filter. Starting from 1 mL spiked whole blood, we recovered 32 ± 4% of the bacteria (Escherichia coli, Klebsiella pneumonia, or Staphylococcus aureus) within one hour while removing 99.4 ± 0.1% of the red blood cells, 98.4 ± 1.4% of the white blood cells, and 90.0 ± 2.6% of the platelets. Changing the device material could further increase bacteria recovery to >50%. We demonstrated bacterial recovery from blood spiked with 10 CFU mL-1. Our simple hands-off efficient separation of low-abundant bacteria approaches clinical expectations, making the new method a promising candidate for future clinical use.

Rapid and label-free Listeria monocytogenes detection based on stimuli-responsive alginate-platinum thiomer nanobrushes.

In this work, we demonstrate the development of a rapid and label-free electrochemical biosensor to detect Listeria monocytogenes using a novel stimulus-response thiomer nanobrush material. Nanobrushes were developed via one-step simultaneous co-deposition of nanoplatinum (Pt) and alginate thiomers (ALG-thiomer). ALG-thiomer/Pt nanobrush platform significantly increased the average electroactive surface area of electrodes by 7 folds and maintained the actuation properties (pH-stimulated osmotic swelling) of the alginate. Dielectric behavior during brush actuation was characterized with positively, neutral, and negatively charged redox probes above and below the isoelectric point of alginate, indicating ALG-thiomer surface charge plays an important role in signal acquisition. The ALG-thiomer platform was biofunctionalized with an aptamer selective for the internalin A protein on Listeria for biosensing applications. Aptamer loading was optimized and various cell capture strategies were investigated (brush extended versus collapsed). Maximum cell capture occurs when the ALG-thiomer/aptamer is in the extended conformation (pH > 3.5), followed by impedance measurement in the collapsed conformation (pH < 3.5). Low concentrations of bacteria (5CFUmL-1) were sensed from a complex food matrix (chicken broth) and selectivity testing against other Gram-positive bacteria (Staphylococcus aureus) indicate the aptamer affinity is maintained, even at these pH values. The new hybrid soft material is among the most efficient and fastest (17min) for L. monocytogenes biosensing to date, and does not require sample pretreatment, constituting a promising new material platform for sensing small molecules or cells.

Inactivation of antibiotic resistant bacteria from stormwater runoff using UVA/LED and its potential risks.

Recently, increasing attention has been paid to antibiotic resistance in stormwater runoff. However, there is no available literature about the control of antibiotic resistant bacteria (ARB) through 365 nm ultraviolet light-emitting diode (UVA/LED). In this study, batch experiments were conducted to investigate ARB inactivation kinetics, effects of light intensity and water matrix (including suspended solid (SS) concentration, initial pH and bacteria concentration), and potential transmission risks after UVA/LED irradiation. Results showed that ARB inactivation efficiencies reached 6.31 log reduction at 8 mW/cm2 (86 J/cm2) of UVA/LED for 180 min. ARB inactivation efficiencies increased with the increase of light intensity, and showed a linear relationship. ARB inactivation decreased with increasing SS levels, and the largest inactivation efficiencies was 3.56 log reduction at 50 mg/L of SS. Initial pH had slight effect on ARB inactivation through UVA/LED irradiation. A low initial bacteria concentration (105 CFU/mL) was not necessarily associated with good ARB inactivation (3.59 log reduction). After UVA/LED irradiation, ARB was hardly detected during 12 hr of dark repair, and the transfer frequency of kanamycin resistance gene was increased to 5.43 × 10-4. These suggested that the application of UVA/LED to inactivate ARB in stormwater runoff was feasible and desirable in this study.

Nanopore Sequencing Using the Full-Length 16S rRNA Gene for Detection of Blood-Borne Bacteria in Dogs Reveals a Novel Species of Hemotropic Mycoplasma.

Dogs across the globe are afflicted by diverse blood- and vector-borne bacteria (VBB), many of which cause severe disease and can be fatal. Diagnosis of VBB infections can be challenging due to the low concentration of bacteria in the blood, the frequent occurrence of coinfections, and the wide range of known, emerging, and potentially novel VBB species encounterable. Therefore, there is a need for diagnostics that address these challenges by being both sensitive and capable of detecting all VBB simultaneously. We detail the first employment of a nanopore-based sequencing methodology conducted on the Oxford Nanopore Technologies (ONT) MinION device to accurately elucidate the "hemobacteriome" from canine blood through sequencing of the full-length 16S rRNA gene. We detected a diverse range of important canine VBB, including Ehrlichia canis, Anaplasma platys, Mycoplasma haemocanis, Bartonella clarridgeiae, "Candidatus Mycoplasma haematoparvum", a novel species of hemotropic mycoplasma, and Wolbachia endosymbionts of filarial worms, indicative of filariasis. Our nanopore-based protocol was equivalent in sensitivity to both quantitative PCR (qPCR) and Illumina sequencing when benchmarked against these methods, achieving high agreement as defined by the kappa statistics (k > 0.81) for three key VBB. Utilizing the ability of the ONT' MinION device to sequence long read lengths provides an excellent alternative diagnostic method by which the hemobacteriome can be accurately characterized to the species level in a way previously unachievable using short reads. We envision our method to be translatable to multiple contexts, such as the detection of VBB in other vertebrate hosts, including humans, while the small size of the MinION device is highly amenable to field use. IMPORTANCE Blood- and vector-borne bacteria (VBB) can cause severe pathology and even be lethal for dogs in many regions across the globe. Accurate characterization of all the bacterial pathogens infecting a canine host is critical, as coinfections are common and emerging and novel pathogens that may go undetected by traditional diagnostics frequently arise. Deep sequencing using devices from Oxford Nanopore Technologies (ONT) provides a solution, as the long read lengths achievable provide species-level taxonomic identification of pathogens that previous short-read technologies could not accomplish. We developed a protocol using ONT' MinION sequencer to accurately detect and classify a wide spectrum of VBB from canine blood at a sensitivity comparable to that of regularly used diagnostics, such as qPCR. This protocol demonstrates great potential for use in biosurveillance and biosecurity operations for the detection of VBB in a range of vertebrate hosts, while the MinION sequencer's portability allows this method to be used easily in the field.

Seasonal Variation of the Atmospheric Bacterial Community in the Greenlandic High Arctic Is Influenced by Weather Events and Local and Distant Sources.

The Arctic is a hot spot for climate change with potentially large consequences on a global scale. Aerosols, including bioaerosols, are important players in regulating the heat balance through direct interaction with sunlight and indirectly, through inducing cloud formation. Airborne bacteria are the major bioaerosols with some species producing the most potent ice nucleating compounds known, which are implicated in the formation of ice in clouds. Little is known about the numbers and dynamics of airborne bacteria in the Arctic and even less about their seasonal variability. We collected aerosol samples and wet deposition samples in spring 2015 and summer 2016, at the Villum Research Station in Northeast Greenland. We used amplicon sequencing and qPCR targeting the 16S rRNA genes to assess the quantities and composition of the DNA and cDNA-level bacterial community. We found a clear seasonal variation in the atmospheric bacterial community, which is likely due to variable sources and meteorology. In early spring, the atmospheric bacterial community was dominated by taxa originating from temperate and Subarctic regions and arriving at the sampling site through long-range transport. We observed an efficient washout of the aerosolized bacterial cells during a snowstorm, which was followed by very low concentrations of bacteria in the atmosphere during the consecutive 4 weeks. We suggest that this is because in late spring, the long-range transport ceased, and the local sources which comprised only of ice and snow surfaces were weak resulting in low bacterial concentrations. This was supported by observed changes in the chemical composition of aerosols. In summer, the air bacterial community was confined to local sources such as soil, plant material and melting sea-ice. Aerosolized and deposited Cyanobacteria in spring had a high activity potential, implying their activity in the atmosphere or in surface snow. Overall, we show how the composition of bacterial aerosols in the high Arctic varies on a seasonal scale, identify their potential sources, demonstrate how their community sizes varies in time, investigate their diversity and determine their activity potential during and post Arctic haze.

Inactivation of Escherichia coli in droplets at different ambient relative humidities: Effects of phase transition, solute and cell concentrations

Previous studies have indicated that ambient relative humidity (RH) plays an important role in the inactivation of bacteria and viruses in droplets. However, characteristics of the RH-dependent inactivation between bacteria and viruses have been reported to differ. Furthermore, how the droplet medium and cell concentration influence inactivation remains unclear. In this study, inactivation of the model bacterium Escherichia coli in droplets of Luria-Bertani (LB) broth and artificial saliva (AS) as a function of RH and initial cell concentration was investigated in a flow cell under a well-controlled environment. Phase transition of aqueous droplets of the two media was observed to occur at ∼50% RH, and their hygroscopicity at different RH were similar. At an initial cell concentration of 1010 CFU/mL, relative viability (RV) in AS droplets showed a V-shape dependence on RH with a minimum at 60%, while RV in LB droplets showed a gradual decrease with decreasing RH. As the initial cell concentration decreased from 1010 to 108 CFU/mL in LB droplets, RV prominently decreased, especially at moderate RH (∼60%), with a V-shape dependence on RH at the lower cell concentration. A drop in RV was also found in AS droplets at a lower initial cell concentration, to below the detection limit in some cases. By considering the bacteria-to-water volume ratio, a high cell concentration in droplets was found to reduce inactivation due to the osmotic stresses. We conclude that for freshly emitted saliva droplets containing a low concentration of bacteria, an ambient RH of ∼60% is optimal for natural inactivation.

DNA Dye Sytox Green in Detection of Bacteriolytic Activity: High Speed, Precision and Sensitivity Demonstrated With Endolysins.

Introduction: Increasing number of deaths from multi-drug resistant bacterial infections has caused both the World Health Organization and the Centers for Disease Control and Prevention to repeatedly call for development of new, non-traditional antibacterial treatments. Antimicrobial enzymes, including those derived from bacteriophages, known as endolysins or enzybiotics, are considered promising solutions among the emerging therapies. These naturally occurring proteins specifically destroy bacterial cell walls (peptidoglycan) and as such, are capable of killing several logs of bacteria within minutes. Some endolysins cause lysis of a wide range of susceptible bacteria, including both Gram-positive and Gram-negative organisms, whereas other endolysins are species- or even strain-specific. To make wide use of endolysins as antibacterial agents, some basic research issues remain to be clarified or addressed. Currently available methods for testing endolysin kinetics are indirect, require large numbers of bacteria, long incubation times and are affected by technical problems or limited reproducibility. Also, available methods are focused more on enzymatic activity rather than killing efficiency which is more relevant from a medical perspective.Results: We show a novel application of a DNA dye, SYTOX Green. It can be applied in comprehensive, real-time and rapid measurement of killing efficiency, lytic activity, and susceptibility of a bacterial population to lytic enzymes. Use of DNA dyes shows improved reaction times, higher sensitivity in low concentrations of bacteria, and independence of bacterial growth. Our data show high precision in lytic activity and enzyme efficiency measurements. This solution opens the way to the development of new, high throughput, precise measurements and tests in variety of conditions, thus unlocking new possibilities in development of novel antimicrobials and analysis of bacterial samples.

Gold-silver core-shell nanodumbbells in solution state as a highly sensitive and reproducible assay platform for bacterial genome detection

Surface-enhanced Raman scattering (SERS) has been investigated as a promising spectroscopic tool for various assay platforms. However, key challenges, such as the reproducibility and sensitivity of this tool should be addressed to enable it to become a reliable and practical method for future clinical applications. Although the single-molecule sensitivity of SERS has already been proven in dry state analysis, its signal reproducibility remains a key challenge in developing a real assay platform. The signal reproducibility of SERS can be significantly improved by obtaining the Raman signal from the solution state; however, the sensitivity of this method is not satisfactory. In this study, we demonstrate a method to overcome both key challenges by using a solution-state formation of gold-silver core-shell nanodumbbells (GSNDs) with target nucleic acid and nanoscale Ag shell formation, which was proven to exhibit single-molecule sensitivity in dry state analysis. To utilize the concept of a reliable assay platform in solution, diverse parameters such as the length of the target sequences, hybridization conditions, number density of probe sequences, and the Ag shell thickness are investigated. The hybridization yield of Au dimer reaches up to 79.36% for E. faecalis, indicating that the hybridization yields are target sequence dependent. It is strongly correlated with detection limit for the target bacteria. The solution-state GSND-based assay shows a highly sensitive results for bacterial target DNA with no mutual interference and excellent reproducibility. The method can identify extremely low concentrations of bacteria (4.5 cfu mL−1), which is much superior to the conventional PCR (4500 cfu mL−1) and culture-based assays (45 cfu mL−1). The results envision the current GSND-based assay as a promising assay platform for future clinical applications.

Farm Silage Facilities and Their Management for the Prevention of Anaerobic Bacteria Spore Contamination in Raw Milk

At feed-out, aerobic spoilage of silage enables an increase in anaerobic spore-forming bacteria (ANSB) that may enter the total mixed ration (TMR). The aim of our study was to understand whether in hot summers the silage structures and management may affect the level of ANSB in milk for long-ripening cheese production. A survey of silage facilities, management, and their relationships with silage, TMR, feces, and milk ANSB most probable number (MPN) content was conducted in the Po Valley during summer months. Silo type did not affect the mean ANSB, but only the wideness of their value distributions, with a narrow range for bags and a wider range for bunkers. The unloading equipment affected the ANSB count; the front-end loader with cutter was associated with a lower ANSB count—probably as a result of the reduced surface left after daily silage removal. Silo length and daily removed face width were the main factors affecting contamination of silage by spore-forming bacteria during summer, with longer silos and wider surface removal reducing ANSB contamination—probably as a consequence of reduced aerobic spoilage at the silage surface. The silage contamination by spore-forming bacteria within a log10 2 MPN g−1 allowed a low concentration of spore-forming bacteria at the farm bulk milk tank level. Fecal ANSB levels did not factor into the regression that explains the ANSB in farm milk. It has been found that silage facilities’ features and their management are an important first step to reduce the extent of ANSB contamination at the farm level.

PM 7/121 (2) ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’

Specific scopeThis Standard describes a diagnostic protocol for ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’.1This Standard should be used in conjunction with PM 7/76 (5) Use of EPPO diagnostic protocols.Specific approval and amendmentFirst approved in 2014–09. Revised in 2021–04.

Isolation of bacteria from artificial bronchoalveolar lavage fluid using density gradient centrifugation and their accessibility by Raman spectroscopy

Raman spectroscopy is an analytical method to identify medical samples of bacteria. Because Raman spectroscopy detects the biochemical properties of a cell, there are many factors that can influence and modify the Raman spectra of bacteria. One possible influence is a proper method for isolation of the bacteria. Medical samples in particular never occur in purified form, so a Raman-compatible isolation method is needed which does not affect the bacteria and thus the resulting spectra. In this study, we present a Raman-compatible method for isolation of bacteria from bronchoalveolar lavage (BAL) fluid using density gradient centrifugation. In addition to measuring the bacteria from a patient sample, the yield and the spectral influence of the isolation on the bacteria were investigated. Bacteria isolated from BAL fluid show additional peaks in comparison to pure culture bacteria, which can be attributed to components in the BAL sample. The isolation gradient itself has no effect on the spectra, and with a yield of 63% and 78%, the method is suitable for isolation of low concentrations of bacteria from a complex matrix.Graphical abstract

Rapid bacteria-detection platform based on magnetophoretic concentration, dielectrophoretic separation, and impedimetric detection

Most biosensors employ small sample quantities (less than 100 μL) for bacteria detection, thereby resulting in inaccurate low-concentration measurements. Detection performed using small sample volumes with low bacteria concentration may produce false-negative results. Therefore, sample pretreatment plays a critical role in accurate bacteria detection. This paper presents an impedimetric bacteria-detection sensor integrated with bacteria concentration and separation devices for rapid bacteria detection. Post conjugation using magnetic particles (MPs), the MP-conjugated bacteria (MP/Bac) are concentrated via magnetophoresis by a factor exceeding 100. In addition, MP/Bac are separated from MPs via dielectrophoresis to prevent occurrence of signal errors caused by MPs not conjugated with bacteria. Subsequently, concentrated MP/Bac are captured on a sensor electrode, and bacteria concentration is detected by measuring signal changes caused by the impedance difference between bacteria and the medium. The performance of the proposed bacteria-detection device was evaluated using a 5-mL homogenized cabbage sample injected with Staphylococcus aureus at 30 mL/h flow rate. The observed signal change was measured for 10 min using a sample with a concentration of 5–5 × 103 CFU/mL and was found to be approximately 0.34 mV at 50 CFU/mL; the limit of detection was 36 CFU/mL. These results confirm that the proposed device can detect low bacteria concentrations in food samples.