Pre-enriched Samples Research Articles

Insights into the degradation of diesel-contaminated seawater through natural microbiota and culturable microorganisms: The overlook role of extracellular polymeric substances (EPS)

The role of EPS produced during biodegradation diesel-contaminated seawater, as well as the effect of EPS on natural microbiota and culturable microorganisms, remains unknown. Here, natural microbiota (pre and post enrichment with diesel) from 5 islands’ seawater in Jiaozhou Bay were explored through metagenomic and culture-dependent approaches. EPS response degradation was mainly characterized by the following aspects: 1) The post-enrichment samples contained significantly fewer dominant bacterial genera than the pre-enrichment samples, and EPS or PS (polysaccharides) promoted degradation of C16-C24 hydrocarbon fractions; 2) EPS caused higher increases in aromatic compound degradation functional genes copies; 3) EPS enhanced the enzymatic activity of alkane hydroxylase and ethanol dehydrogenase; 4) Diesel degradation by mono and mixed cultures of culturable bacteria (B1 and B2) with extracted EPS showed a higher biomass, biosurfactant production, expression of PAH-RHDα GN and cytochrome P450 genes, degradability and emulsification capacity. The above innovations provided new perspectives for oil-spill bioremediation.

Comparative study on the effect of bacteriophage on MDR E.coli from Urinary tract infections

Abstract Introduction/Objective Background: Urinary tract infections (UTIs) associated with urethral catheters are the most common infections during hospitalization. Hospitalization's most common infections are urethral catheter-related UTIs. Bacteriophages infect bacteria exclusively. Phage therapy, especially in Eastern Europe, was widely used before antibiotics. Due to the overuse of antibiotics and the development of resistant bacteria, this therapy approach is regaining popularity. Methods/Case Report Subjects and methods: 2223 UAE-based UTI patients (4–68 years old) were examined. The isolated bacteria were identified biochemically and tested for antimicrobial susceptibility using Mueller-Hinton agar disk diffusion (Kirby Bauer's). TSA (Liofilchem, Italy) kept isolates at 4 °C.for each examination. Tryptic Soy agar (TSA; Liofilchem, Italy) was used to grow one isolated colony overnight at 37 °C. This culture was transferred aseptically to 10 mL of fresh TSB medium and grown overnight at 37 °C to 0.8 optical density. 10 ml CaCl2 in 100 ml LB homogenized EMIRATE sewage water. Overnight homogenates with 9 E. coli isolates—24 hours of 37°C incubation. Samples were centrifuged at 12,000x g for five minutes—a 0.45 mm Millipore (France) filter produced phage lysate. TSA (Liofilchem, Italy) was used as a culture medium, and the centrifuged supernatant as phage suspension for the double-layer method. After 37 °C, plaques were examined. Single-plaque isolations followed. EOP was measured on double-layer agar for bacteria with a clear lysis zone (Adams, 1959). calculated EOP. Phages tested individually. EZ1 and EZ2 were negatively stained with Na-phosphotungstate for morphotype. Results (if a Case Study enter NA) Results: This study found presumptive E. coli colonies in 478 of 2223 (21.5%) samples by plating pre-enriched samples on MacConkey. E. coli B2 and B4 were sensitive to Ciprofloxacin, but most strains were resistant to antibiotics. E. coli B3 was Metronidazole-sensitive. Individual E. coli strains were susceptible to phage infection. Podoviruses were the two phages. 3 log decreased due to E. coli growth. Conclusion This study shows that a single dose of phage can reduce pathogenic MDR E.coli. Phages' high bacterial inactivation efficiency, safety, and prolonged survival, even in urine samples, allow depth studies, especially in vivo studies, to control urinary tract infection and overcome E.coli resistance.

Growth assessment of Salmonella enterica multi-serovar populations in poultry rinsates with commonly used enrichment and plating media

Isolation of Salmonella from enrichment cultures of food or environmental samples is a complicated process. Numerous factors including fitness in various selective enrichment media, relative starting concentrations in pre-enrichment, and competition among multi-serovar populations and associated natural microflora, come together to determine which serovars are identified from a given sample. A recently developed approach for assessing the relative abundance (RA) of multi-serovar Salmonella populations (CRISPR-SeroSeq or Deep Serotyping, DST) is providing new insight into how these factors impact the serovars observed, especially when different selective enrichment methods are used to identify Salmonella from a primary enrichment sample. To illustrate this, we examined Salmonella-positive poultry pre-enrichment samples through the selective enrichment process in Tetrathionate (TT) and Rappaport Vassiliadis (RVS) broths and assessed recovery of serovars with each medium. We observed the RA of serovars detected post selective enrichment varied depending on the medium used, initial concentration, and competitive fitness factors, all which could result in minority serovars in pre-enrichment becoming dominant serovars post selective enrichment. The data presented provide a greater understanding of culture biases and lays the groundwork for investigations into robust enrichment and plating media combinations for detecting Salmonella serovars of greater concern for human health.

Detection of blaCTX-M and blaDHA genes in stool samples of healthy people: comparison of culture- and shotgun metagenomic-based approaches.

We implemented culture- and shotgun metagenomic sequencing (SMS)-based methods to assess the gut colonization with extended-spectrum cephalosporin-resistant Enterobacterales (ESC-R-Ent) in 42 volunteers. Both methods were performed using native and pre-enriched (broth supplemented with cefuroxime) stools. Native culture screening on CHROMID® ESBL plates resulted in 17 positive samples, whereas the pre-enriched culture (gold-standard) identified 23 carriers. Overall, 26 ESC-R-Ent strains (24 Escherichia coli) were identified: 25 CTX-M and 3 DHA-1 producers (2 co-producing CTX-Ms). Using the SMS on native stool ("native SMS") with thresholds ≥60% for both identity and coverage, only 7 of the 23 pre-enriched culture-positive samples resulted positive for blaCTX-M/blaDHA genes (native SMS reads mapping to blaCTX-M/blaDHAs identified in gold-standard: sensitivity, 59.0%; specificity 100%). Moreover, an average of 31.5 and 24.6 antimicrobial resistance genes (ARGs) were detected in the 23 pre-enriched culture-positive and the 19 negative samples, respectively. When the pre-enriched SMS was implemented, more blaCTX-M/blaDHA genes were detected than in the native assay, including in stools that were pre-enriched culture-negative (pre-enriched SMS reads mapping to blaCTX-M/blaDHAs identified in gold-standard: sensitivity, 78.3%; specificity 75.0%). In addition, the pre-enriched SMS identified on average 38.6 ARGs/sample, whereas for the corresponding native SMS it was 29.4 ARGs/sample. Notably, stools resulting false-negative by using the native SMS had lower concentrations of ESC-R-Ent (average: ~105 vs. ~107 CFU/g) and E. coli classified reads (average: 193,959 vs. 1.45 million) than those of native SMS positive samples. Finally, the detection of blaCTX-M/blaDHA genes was compared with two well-established bioinformatic tools. In conclusion, only the pre-enriched SMS assured detection of most carriers of ESC-R-Ent. However, its performance was not comparable to the pre-enriched culture-based approach.

Determination of Bacterial Diversity of Propolis Microbiota.

Propolis is a natural resinous mixture produced by the excretions of honeybees. PCR amplification of the 16S rRNA gene region was achieved using DNA of pre-enriched propolis samples collected from Apis mellifera production hives (n=37) in Eastern Türkiye (Bingöl and its regions). Next-generation sequencing and metabarcoding techniques were used to identify bacterial communities in propolis samples. Firmicutes dominated the phylum structure, with Proteobacteria, Actinobacteria, Tenericutes, and Spirochaetes following. The top three bacterial families were Bacillaceae, Enterobacteriaceae, and Enterococcaceae. Bacillus (dominantly B. badius and B. thermolactis at the species level) was recognized at the genus level, followed by Enterococcus and Clostridium sensu stricto. Our study comprehensively identified the bacterial diversity of propolis samples. Further investigations targeting to enlighten the microbiota of propolis and its potential application fields are required to gain better insight into ecological, nutritional, and medicinal perspectives.

Determination of bacterial community structure of Turkish kefir beverages via metagenomic approach

Bacterial microbiota of industrially produced kefir beverages (n:33) consumed in Turkey was studied using a culture-independent method and a metagenomic approach. DNA extraction from non pre-enriched and pre-enriched kefir samples was used for 16S rRNA amplicon sequencing. Kefirs were dominated by Firmicutes, followed by Actinobacteria and Proteobacteria phyla. The most abundant genera in non pre-enriched kefir beverages were Lactococcus followed by Streptococcus, Bifidobacterium, Lactobacillus, and Leuconostoc. Pre-enriched kefirs were dominated by Streptococcus followed by Lactobacillus, Lactococcus, Bifidobacterium, and Leuconostoc at the genus level. Psychroserpens, Desulfonispora, Pediococcus, Micromonospora, Fructobacillus, Mycobacterium, Acetobacter, Pseudopedobacter, and Clostridium XI genera were found only in pre-enriched kefirs. Kefirs displayed pH differences from 4.04 to 4.49 and the acidity was 0.617–0.987. In two samples, the lowest pH values were obtained with abundance of Lactobacillus helveticus and Streptococcus salivarius. This study broadens our viewpoint and strengthens future applications of kefir beverages in industrial and medical fields.

Microbial community of soda Lake Van as obtained from direct and enriched water, sediment and fish samples

Soda lakes are saline and alkaline ecosystems that are considered to have existed since the first geological records of the world. These lakes support the growth of ecologically and economically important microorganisms due to their unique geochemistry. Microbiota members of lakes are valuable models to study the link between community structure and abiotic parameters such as pH and salinity. Lake Van is the largest endroheic lake and in this study, bacterial diversity of lake water, sediment, and pearl mullet (inci kefali; Alburnus tarichi), an endemic species of fish which are collected from different points of the lake, are studied directly and investigated meticulously using a metabarcoding approach after pre-enrichment. Bacterial community structures were identified using Next Generation Sequencing of the 16S rRNA gene. The analysis revealed that the samples of Lake Van contain high level of bacterial diversity. Direct water samples were dominated by Proteobacteria, Cyanobacteria, and Bacteroidota, on the other hand, pre-enriched water samples were dominated by Proteobacteria and Firmicutes at phylum-level. In direct sediment samples Proteobacteria, whereas in pre-enriched sediment samples Firmicutes and Proteobacteria were determined at highest level. Pre-enriched fish samples were dominated by Proteobacteria and Firmicutes at phylum-level. In this study, microbiota members of Lake Van were identified by taxonomic analysis.

Identification of bacterial communities of fermented cereal beverage Boza by metagenomic analysis

Bacterial microbiota of directly studied and pre-enriched Boza samples were investigated by metagenomic analysis. Virulence gene contents, biofilm formation, antibiotic susceptibility and clonal relationships of enterococci present in pre-enriched Boza samples were determined. Chemical properties of the samples were also investigated. Although directly studied samples showed a dominance by Lactococcus, Lactobacillus, Leuconostoc, and Streptococcus. NGS upon pre-enrichment of the same Boza samples demonstrated a dominance by Lactococcus, Enterococcus, Escherichia/Shigella, Bacillus, and Lactobacillus. All enterococci were identified as Enterococcus faecium and none of them was positive for vanA, vanB, vanC1, vanD, vanE, vanG, agg, gelE, efaAfs, cylA, ace, hyl, cob, cylB, and cylM genes. However, efaAfm, ccf, cpd, and esp genes were detected in the strains. Only one strain formed biofilm and seven strains showed low adherence. E. faecium strains were resistant to rifampin and erythromycin. PFGE revealed 54–100% clonal relationships of E. faecium strains. Percent acidity of Boza samples were 0.14%–0.51%, pH was 3.00–4.07, protein content was 0.35–1.23 mg/100 mg, total sugar content was 9.64–19.21 mg/100 mg Boza, crude ash content was 0.05–0.18 mg/100 mg dry sample, total dry matter was 13.79–28.04 mg/100 mg. Our results indicate to importance of the dynamics nature of microbial communities involved in Boza fermentation and virulence properties of enterococci.

Comparative occurrence and antibiogram of extended-spectrum β-lactamase-producing Escherichia coli among post-weaned calves and lactating cows from smallholder dairy farms in a parallel animal husbandry area.

Background and Aim:Inappropriate overuse of antimicrobials might be associated with the spreading of antimicrobial-resistant bacteria in animal-based food products. Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli have been recognized as an emerging global problem in a One Health approach. This study aimed to assess the occurrence and antimicrobial-susceptible profiles of ESBL-producing E. coli among post-weaned calves and lactating cows in a parallel animal husbandry area.Materials and Methods:Seventy-two pool fecal samples were collected from 36 smallholder dairy farms registered in Ban Hong Dairy Cooperatives, Lamphun Province, Thailand. Pre-enriched fecal samples were cultured in MacConkey agar supplemented with cefotaxime. The potential E. coli isolates were identified by not only biochemical tests but also polymerase chain reaction assay of the 16S rRNA gene. ESBL production was confirmed by the combination disk test. Antimicrobial susceptibility testing was performed by the Kirby–Bauer disk diffusion method.Results:The occurrence of ESBL-producing E. coli at the farm level was 80.56%. The different phenotypic antibiogram of ESBL-producing E. coli was observed among post-weaned calf and lactating cow specimens. The most frequent resistance patterns of ESBL-producing isolates from both groups were amoxicillin-ceftiofur-cephalexin-cephalothin-cloxacillin-streptomycin-oxytetracycline-sulfamethoxazole/trimethoprim. For the median zone diameter, enrofloxacin-resistant isolates with narrow zone diameter values from lactating cow specimens were particularly more than post-weaned calf specimens (p<0.05).Conclusion:These findings revealed the dynamic changes in ESBL-producing E. coli from calves and lactating cows in Lamphun Province, posing the inevitability to prevent bacterial transmission and optimize antimicrobial therapy in dairy farming.

Effect of natural microbiome and culturable biosurfactants-producing bacterial consortia of freshwater lake on petroleum-hydrocarbon degradation

Freshwater lake ecosystem is a reservior of valuable microbial diversity. It needs to be explored for addressing key environmental issues like petroleum-hydrocarbon contamination. In this work, the microbial communities (pre and post enriched with petroleum-hydrocarbons) from different layers of freshwater lake, i.e. surface water, sediments and deepwater, were explored through metagenomic and culture-dependent approaches. A total of 41 bacterial phyla were retrieved from pre-enriched samples, which were significantly reduced in enriched samples where Proteobacteria were dominant (87% to 100%) followed by Bacteroidetes (7.37%) and Verrucomicrobia (3.06%). The most dominant hydrocarbon-degrading genera were extensively verified as Pseudomonas (48.65%), Acinetobacter (45.38%), Stenotrophomonas (3.16%) and Brevundimonas (2.07%) in surface water (S1WCC); Acinetobacter (62.46%), Aeromonas (10.7%), Sphingobacterium (5.20%) and Pseudomonas (4.23%) in sediment (S2MCC); and Acinetobacter (46.57%), Pseudomonas (13.10%), Comamonas (12.93%), Flavobacterium (12.18%) and Enterobacter (9.62%) in deep water (S4WCC). Additionally, the maximum biodegradation of petroleum-hydrocarbons (i.e. used engine oil or UEO) was achieved by microbiome of S2MCC (67.60 ± 0.08%) followed by S4WCC (59.70 ± 0.12%), whereas only 36.80 ± 0.10% degradation was achieved by S1WCC microbiome. On the other hand, UEO degradation by cultivable biosurfactant-producing single cultures such as Pseudomonas sp. S2WE, Pseudomonas sp. S2WG, Pseudomonas sp. S2MS, Ochrobactrum sp. S1MM and Bacillus nealsonii S2MT showed 31.10 ± 0.08% to 40.50 ± 0.11% biodegradation. Comparatively, the biodegradation efficiency was found higher (i.e. 42.20 ± 0.12% to 56.10 ± 0.12%) in each consortia comprising of two, three, four, and five bacterial cultures. Conclusively, the isolated culturable biosurfactants-producing bacterial consortium of freshwater lake demonstrated >80% contribution in the total petroleum-hydrocarbons degradation by the natural microbiome of the ecosystem.

Evaluation of Microfluidic Ceiling Designs for the Capture of Circulating Tumor Cells on a Microarray Platform

The capture of circulating tumor cells (CTCs) is still a challenging application for microfluidic chips, as these cells are rare and hidden in a huge background of blood cells. Here, different microfluidic ceiling designs in regard to their capture efficiency for CTCs in model experiments and more realistic conditions of blood samples spiked with a clinically relevant amount of tumor cells are evaluated. An optimized design for the capture platform that allows highly efficient recovery of CTCs from size-based pre-enriched samples under realistic conditions is obtained. Furthermore, the viability of captured tumor cells as well as single cell recovery for downstream genomic analysis is demonstrated. Additionally, the authors' findings underline the importance of evaluating rational design rules for microfluidic devices based on theoretical models by application-specific experiments.

Population Analyses Reveal Preenrichment Method and Selective Enrichment Media Affect Salmonella Serovars Detected on Broiler Carcasses

Poultry is a major Salmonella reservoir, but conventional culture-based methods typically identify the most abundant serovars while those less abundant remain undetected. Choice of enrichment procedure also introduces bias, and for broiler carcasses, a 1-min rinse before preenrichment is insufficient to release all Salmonella present. The inability to assess serovar diversity means that serovars more often associated with human illness may be masked by more abundant Salmonella. CRISPR-SeroSeq (serotyping by sequencing clustered regularly interspaced short palindromic repeats), an amplicon-based, next-generation sequencing tool, allows detection of multiple serovars and maps the relative serovar frequencies in a sample. To address the preceding limitations, CRISPR-SeroSeq was used on broiler carcasses collected prechilled at a commercial plant. Standard carcass rinse aliquot preenrichments and whole carcass preenrichments that were enriched in Rappaport-Vassiliadis (RV) and tetrathionate (TT) broths were compared. On average, five serovars were observed per carcass, including nine on one carcass. CRISPR-SeroSeq detected serovars comprising as little as 0.005% of the population. CRISPR-SeroSeq data matched (28 of 32) standard culture analysis for abundant serovars. Salmonella serovars Kentucky, Typhimurium, and Schwarzengrund were found on each carcass. Overall, serovar diversity was higher in whole carcass preenrichments that were enriched in RV (P < 0.05). Serovar Schwarzengrund was present at higher frequencies in whole carcass preenrichments compared with rinse aliquot preenrichments (t test, P < 0.05), suggesting it adheres more strongly to the carcass. Salmonella serovar Enteritidis was enriched eightfold more in TT than in RV, and serovars Schwarzengrund and Reading were preferentially enriched in RV. Comparison of preenriched and enriched samples suggests that selective enrichment in RV or TT was inhibitory to some serovars. This article addresses limitations of Salmonella surveillance protocols and provides information related to Salmonella population dynamics.

Molecular detection of Salmonella serovars Enteritidis, Heidelberg and Typhimurium directly from pre-enriched poultry samples

ABSTRACT 1. Salmonella is one of the most important pathogens in public health and it is usually associated with food-borne diseases. Salmonella serovars Enteritidis and Typhimurium are widespread in the world with outbreaks frequently associated with consumption of poultry products; furthermore, there is an increasing public health concern with the wide dissemination of the serovar Heidelberg in poultry flocks. 2. The aim of the experiment was to develop and to validate rapid methods to detect Salmonella serovars Enteritidis, Typhimurium, and Heidelberg by real-time PCRs and test isolates from pre-enriched poultry samples. 3. Three real-time PCRs were developed and used in combination to detect the serovars Enteritidis, Typhimurium and Heidelberg. These assays were validated by the analysis of 126 Salmonella isolates, eight other enteric bacterial species and 34 naturally contaminated poultry samples after pre-enrichment with buffered peptone water (BPW). 4. Real-time PCRs detected the isolates of the most important poultry serovars (Enteritidis, Typhimurium and Heidelberg) with 100% inclusivity and exclusivity in each assay. The PCR identified monophasic variants of the serovars Typhimurium and Heidelberg. All PCRs were validated in detecting these specific serovars directly from pre-enriched poultry samples. The whole analytical procedure was performed in less than 24 h in a veterinary diagnostic laboratory.

A multiplex real-time PCR assay, based on invA and pagC genes, for the detection and quantification of Salmonella enterica from cattle lymph nodes

Cattle lymph nodes can harbor Salmonella and potentially contaminate beef products. We have developed and validated a new real-time PCR (qPCR) assay for the detection and quantification of Salmonella enterica in cattle lymph nodes. The assay targets both the invA and pagC genes, the most conserved molecular targets in Salmonella enterica. An 18S rRNA gene assay that amplifies from cattle and other animal species was also included as an internal control. Available DNA sequences for invA, pagC and 18S rRNA genes were used for primer and probe selections. Three Salmonella serotypes, S. Typhimurium, S. Anatum, and S. Montevideo, were used to assess the assay's analytical sensitivity. Correlation coefficients of standard curves generated for each target and for all three serotypes were >99% and qPCR amplification efficiencies were between 93% and 110%. Assay sensitivity was also determined using standard curve data generated from Salmonella-negative cattle lymph nodes spiked with 10-fold dilutions of the three Salmonella serotypes. Assay specificity was determined using Salmonella culture method, and qPCR testing on 36 Salmonella strains representing 33 serotypes, 38 Salmonella strains of unknown serotypes, 252 E. coli strains representing 40 serogroups, and 31 other bacterial strains representing 18 different species. A collection of 647 cattle lymph node samples from steers procured from the Midwest region of the US were tested by the qPCR, and compared to culture-method of detection. Salmonella prevalence by qPCR for pre-enriched and enriched lymph nodes was 19.8% (128/647) and 94.9% (614/647), respectively. A majority of qPCR positive pre-enriched samples (105/128) were at concentrations between 104 and 105 CFU/mL. Culture method detected Salmonella in 7.7% (50/647) and 80.7% (522/647) of pre- and post-enriched samples, respectively; 96.0% (48/50) of pre-enriched and 99.4% (519/522) of post-enriched culture-positive samples were also positive by qPCR. More samples tested positive by qPCR than by culture method, indicating that the real-time PCR assay was more sensitive. Our data indicate that this triplex qPCR can be used to accurately detect and quantify Salmonella enterica strains from cattle lymph node samples. The assay may serve as a useful tool to monitor the prevalence of Salmonella in beef production systems.

Evaluación de una Técnica de PCR-Múltiple para la Detección Rápida de Salmonella Typhimurium y Enteritidis en Cuyes (Cavia porcellus) Naturalmente Infectados

El objetivo del estudio fue evaluar la capacidad de detección de Salmonella Typhimurium y Enteritidis mediante el método de la Reacción en Cadena de la Polimerasa (PCR) Múltiple a partir de muestras en pre-enriquecimiento no selectivo (utilizando secuencias blanco de los genes InvA, fliC y prot6E), y hallar su concordancia con el método de detección microbiológica convencional, que consta de pre-enriquecimiento no selectivo, enriquecimiento selectivo, aislamiento en agar diferencial y pruebas bioquímicas. Se analizaron 111 muestras de hígado de cuyes con diagnóstico presuntivo de Salmonelosis, provenientes de Chancay (Lima) y El Mantaro (Junín), Perú. Se llegó a detectar Salmonella Typhimurium por PCR Múltiple en 54% (60/111) de las muestras y en 41% (45/111) por análisis microbiológico. Se encontró una concordancia substancial con un valor de Kappa de 0.64. La prueba de McNemar demostró que los resultados de ambas pruebas son estadísticamente diferentes (p&lt;0.05).

Fast and efficient enrichment of functional ILC2 from human whole blood

Abstract Group 2 innate lymphoid cells (ILC2) are a functionally distinct subset of recently identified immune cells with important roles in type-2 immunopathologies such as allergies, asthma, helminth infections and other metabolic diseases. Studying these rare cells is challenging due to a lack of specific surface markers, and currently multicolor flow cytometric analysis and cell sorting are the only methods to characterize and isolate ILC2s. However, the scarcity of these cells makes flow cytometry time-consuming, expensive and often results in low purities and recoveries. Thus, better approaches for effective identification and isolation are essential to further understanding of ILC2 biology and function. We have developed a rapid and efficient method for enrichment of human ILC2 from whole blood. In brief, non-ILC2 cells in whole blood were crosslinked to red blood cells already present in the sample using RosetteSep™. The sample was then layered over Lymphoprep in a SepMate™ tube, spun at 1200 x g for 10 minutes (min), and the untouched, desired cells simply poured off. Cells were washed once and were then ready for subsequent analysis. Starting with only 0.01 – 0.07% in whole blood, ILC2 were enriched 350 ± 220 fold to 8.2 ± 6.8% in 35 min (means ± SD, n=17). Subsequent cell sorting from these pre-enriched samples was faster and yielded higher purity ILC2 than sorting from non-enriched controls (n=3, p&amp;lt;0.05 paired t test). Sorted ILC2, both pre-enriched and non-enriched controls, were cultured and stimulated, and secreted similar high levels of IL-13 as assessed by ELISA, indicating that these cells are functional. In summary, ILC2 pre-enrichment improves sorting efficiency, increases ILC2 purity, and maintains ILC2 functionality.

Burkholderia pseudomallei is frequently detected in groundwater that discharges to major watercourses in northern Australia.

Burkholderia pseudomallei is the environmental bacterium that causes the serious disease melioidosis. Recently, a high prevalence of viable B. pseudomallei was reported from natural groundwater seeps around Castle Hill, a clinical focus of melioidosis in Townsville, Australia. This study sought to expand previous findings to determine the extent of B. pseudomallei in more diverse natural groundwater seeps in northern Queensland to ascertain if the presence of the organism in groundwater on Castle Hill was an isolated occurrence. Analysis of water samples (n = 26) obtained from natural groundwater seeps following an intensive rainfall event in the Townsville region determined the presence of B. pseudomallei DNA in duplicates of 18 samples (69.2% [95% CI, 51.5 to 87.0]). From 26 water samples, a single isolate of B. pseudomallei was recovered despite plating of both pre-enriched samples and original water samples onto selective media, indicating that the sensitivity of these molecular techniques far exceeds culture-based methods. Furthermore, the identification of new environments endemic for melioidosis may be more effectively determined by analysing surface groundwater seeps than by the analysis of random soil samples. This study suggests that a higher incidence of melioidosis following monsoonal rains may be partially the result of exposure to groundwater sources carrying B. pseudomallei, and that modifications to public health messages in endemic regions may be warranted. Moreover, these findings have implications for predictive models of melioidosis, effective models requiring consideration of topographical and surface hydrological data.

Isothermal Target and Probe Amplification Assay for the Real-Time Rapid Detection of Staphylococcus aureus

Staphylococcus aureus, the species most commonly associated with staphylococcal food poisoning, is one of the most prevalent causes of foodborne disease in Korea and other parts of the world, with much damage inflicted to the health of individuals and economic losses estimated at $120 million. To reduce food poisoning outbreaks by implementing prevention methods, rapid detection of S. aureus in foods is essential. Various types of detection methods for S. aureus are available. Although each method has advantages and disadvantages, high levels of sensitivity and specificity are key aspects of a robust detection method. Here, we describe a novel real-time isothermal target and probe amplification (iTPA) method that allows the rapid and simultaneous amplification of target DNA (the S. aureus nuc gene) and a fluorescence resonance energy transfer–based signal probe under isothermal conditions at 61°C or detection of S. aureus in real time. The assay was able to specifically detect all 91 S. aureus strains tested without nonspecific detection of 51 non–S. aureus strains. The real-time iTPA assay detected S. aureus at an initial level of 101 CFU in overnight cultures of preenriched food samples (kiwi dressing, soybean milk, and custard cream). The advantage of this detection system is that it does not require a thermal cycler, reducing the cost of the real-time PCR and its footprint. Combined with a miniaturized fluorescence detector, this system can be developed into a simplified quantitative hand-held real-time device, which is often required. The iTPA assay was highly reliable and therefore may be used as a rapid and sensitive means of identifying S. aureus in foods.

Shiga Toxin-Producing Escherichia coli in Meat and Vegetable Products in Emilia Romagna Region, Years 2012-2013.

In 2012-2013 Emilia-Romagna Region introduced a monitoring plan for Shiga toxin-producing Escherichia coli (STEC) in foodstuff. Six hundred eighty-nine meat samples and 273 fruit and vegetable products were analyzed according to ISOTS13136. Pre-enriched samples were tested by multiplex real time PCR targeting the virulence genes eae, stx1 and stx2. Stx2 positive samples were investigated for the presence of serogroup O104 associated gene. O103, O111, O145, O157, O26 associated genes were tested on samples positive for stx in association with eae gene. Isolation of E. coli strains was attempted from samples positive for serogroup-associated genes. Thirty-four meat products (4.9%) resulted positive for stx1 and/or stx2 genes and 46 (6.7%) for stx1 and/or stx2 genes in association with eae gene. Forty-five (6.5%) samples resulted positive at least at one serogroup. Serogroup O103, O104, O111, O145, O157 and O26 genes were detected respectively in 1.3, 0.3, 0.1, 3.9, 2.9 and 2.5% samples; 0.6% samples resulted positive for STEC isolation (2 E. coli O103 and 2 E. coli O157). It is worth noting that STEC virulence genes were detected at high frequency (19%) in fresh pork meat sausages. Four (1.5%) vegetable samples were positive for stx1 and/or stx2 genes and 1 (0.4%) for stx1 and/or stx2 genes in association with eae gene; none resulted positive for the tested serogroups.Only a low number of samples positive by molecular methods were confirmed by cultural isolation. It is therefore of the uttermost importance for appropriate risk management, to be fully aware of the meaning of the analytical result.

Sample Preparation Methods Following CellSearch Approach Compatible of Single-Cell Whole-Genome Amplification: An Overview.

Single cells are increasingly used to determine the heterogeneity of therapy targets in the genome during the course of a disease. The first challenge using single cells is to isolate these cells from the surrounding cells, especially when the targeted cells are rare. A number of techniques have been developed for this goal, each having specific limitations and possibilities. In this chapter, five of these techniques are discussed in the light of the isolation of circulating tumor cells (CTC) present at extremely low frequency in the blood of patients with metastatic cancer from the perspective of pre-enriched samples by means of CellSearch. The techniques described are micromanipulation, FACS, laser capture microdissection, DEPArray, and microfluidic solutions. All platforms are hampered with a low efficiency and differences in hands-on time and costs are the most important drivers for selection of the optimal platform.