Journal of microbiological methods最新文献

Salmonella enterica serovar Infantis (S. infantis) is an important emerging pathogen, associated with poultry and poultry products and related to an increasing number of human infections in many countries. A concerning trend among S. infantis isolates is the presence of plasmid-mediated multidrug resistance. In many instances, the genes responsible for this resistance are carried on a megaplasmid known as the plasmid of emerging S. infantis (pESI) or pESI like plasmids. Plasmids can be remarkably stable due to the presence of multiple replicons and post-segregational killing systems (PSKs), which contribute to their maintenance within bacterial populations. To enhance our understanding of S. infantis and its multidrug resistance determinants toward the development of new vaccination strategies, we have devised a new method for targeted plasmid curing. This approach effectively overcomes plasmid addiction by leveraging the temporal overproduction of specific antitoxins coupled with the deletion of the partition region. By employing this strategy, we successfully generated a plasmid-free strain from a field isolate derived from S. infantis 119,944.

This method provides valuable tools for studying S. infantis and its plasmid-borne multidrug resistance mechanisms and can be easily adopted for plasmid curing from other related bacteria.

The CDC Biofilm Reactor method is the standard biofilm growth protocol for the validation of US Environmental Protection Agency biofilm label claims. However, no studies have determined the effect of coupon orientation within the reactor on biofilm growth. If positional effects have a statistically significant impact on biofilm density, they should be accounted for in the experimental design. Here, we isolate and quantify biofilms from each possible coupon surface in the reactor to quantitatively determine the positional effects in the CDC Biofilm Reactor. The results showed no statistically significant differences in viable cell density across different orientations and vertical positions in the reactor. Pseudomonas aeruginosa log densities were statistically equivalent among all coupon heights and orientations. While the Staphylococcus aureus cell growth showed no statistically significant differences, the densities were not statistically equivalent among all coupon heights and orientations due to the variability in the data. Structural differences were observed between biofilms on the high-shear baffle side of the reactor compared to the lower shear glass side of the reactor. Further studies are required to determine whether biofilm susceptibility to antimicrobials differs based on structural differences attributed to orientation.

The high prevalence of dental caries and periodontal disease place a significant burden on society, both socially and economically. Recent advances in genomic technologies have linked both diseases to shifts in the oral microbiota – a community of >700 bacterial species that live within the mouth. The development of oral microbiome transplantation draws on the success of fecal microbiome transplantation for the treatment of gut pathologies associated with disease. Many current in vitro oral biofilm models have been developed but do not fully capture the complexity of the oral microbiome which is required for successful OMT. To address this, we developed an in vitro biofilm system that maintained an oral microbiome with 252 species on average over 14 days. Six human plaque samples were grown in 3D printed flow cells on hydroxyapatite discs using artificial saliva medium (ASM). Biofilm composition and growth were monitored by high throughput sequencing and confocal microscopy/SEM, respectively. While a significant drop in bacterial diversity occurred, up to 291 species were maintained in some flow cells over 14 days with 70% viability grown with ASM. This novel in vitro biofilm model represents a marked improvement on existing oral biofilm systems and provides new opportunities to develop oral microbiome transplant therapies.

A novel method for the quantification of antifungal activity of fungicides and painted surfaces, mycelial invasion distance (MID) method, was developed and applied to the quantification of activities of parabens and an antifungal paint. In this method, the MID of aerial mycelia on a test paper or a panel placed on a nutrient agar plate was measured with a stereoscopic microscope and a micro-ruler. The antifungal activities of the parabens and painted surfaces were expressed as the MID. The higher the hydrophobicity of parabens, the longer the MID, that is the lower the antifungal activity, were observed. Conversely, relatively polar parabens, such as methyl and ethyl parabens, exhibited stronger antifungal activity, that is shorter MID. The most hydrophobic paraben, benzyl paraben, showed the weakest antifungal activity. Furthermore, it was confirmed that the MID method was effective for the evaluation of the painted surfaces.

As data accumulate in GenBank, the difficulties of delineating species of Cryptosporidium based on nuclear small subunit ribosomal RNA (ssu rRNA) gene information alone becomes increasingly evident. Here, we summarize currently available evidence suggesting that several ssu rDNA sequences primarily referred to as Cryptosporidium suis (some of them from non-suid hosts) should be considered Cryptosporidium occultus.

Bacterial meningitis is an acute infection which requires rapid diagnosis and treatment due to the high mortality and serious consequences of the disease. The purpose of this study was to design a homemade multiplex PCR and a novel fluorescence biosensor on chip (FBC) to detect three important agents of meningitis including Streptococcus pneumoniae (S. pneumoniae), Neisseria meningitidis (N. meningitidis), and Haemophilus influenzae (H. influenzae). The homemade multiplex PCR can diagnose three bacterial species simultaneously. Fabrication of FBC was carried out based on the deposition of lead nanoparticles on a quartz slide using the thermal evaporation method. Then, the SH-Cap Probe/Target ssDNA /FAM-Rep probe was loaded on lead film. The evaluation of the fluorescence reaction when the probes bind to the target ssDNA was assessed by a Cytation 5 Cell Imaging Multimode Reader Bio-Tek. The limit of detections (LOD) in homemade PCR and FBC to identify S. pneumoniae were 119 × 10² CFU/mL (0.27 ng/μL) and 380 CFU/mL (9 pg/μL), respectively. The LODs of homemade PCR and FBC for detection of N. meningitidis were 4.49 CFU/mL (1.1 pg/μL) and 13 × 10³ CFU/mL (30 pg/μL), respectively. Our results confirmed the LODs of homemade PCR and FBC in detection of H. influenzae were 15.1 CFU/mL (30 fg/μL) and 41 × 10² CFU/mL (90 pg/ μL), respectively. Both techniques had appropriate sensitivity and specificity in detection of S. pneumoniae, N. meningitidis and H. influenzae.

Flow cytometry (FCM) provides unique information on bacterial viability and physiology, allowing a real-time early warning antimicrobial and antibiofilm monitoring system for preventing the spread risk of foodborne disease. The present work used a combined culture-based and FCM approach to assess the in vitro efficacy of essential oils (EOs) from condiment plants commonly used in Mediterranean Europe (i.e., thyme EO, oregano EO, basil EO, and lemon EO) against planktonic and sessile cells of food-pathogenic Listeria monocytogenes 56 LY, and contaminant and alterative species Escherichia coli ATCC 25922 and Pseudomonas fluorescens ATCC 13525. Evaluation of the bacterial response to the increasing concentrations of natural compounds posed FCM as a crucial technique for the quantification of the live/dead, and viable but non-culturable (VBNC) cells when antimicrobial agents exert no real bactericidal action. Furthermore, the FCM results displayed higher numbers of viable bacteria expressed as Active Fluorescent Units (AFUs) with a greater level of repeatability compared with outcomes of the plate-count method. Overall, accurate counting of viable microbial cells is a critically important parameter in food microbiology, and flow cytometry provides an innovative approach with high-throughput potential for applications in the food industry as “flow microbiology”.

We aim to objectify the evaluation criteria of agglutination rate estimation in the Microscopic Agglutination Test (MAT). This study proposes a deep learning method that extracts free leptospires from dark-field microscopic images and calculates the agglutination rate. The experiments show the effect of objectification with real pictures.

The microbial composition and stress molecules are main drivers influencing the development and spread of antibiotic resistance bacteria (ARBs) and genes (ARGs) in the environment. A reliable and rapid method for identifying associations between microbiome composition and resistome remains challenging. In the present study, secondary metagenome data of sewage and hospital wastewaters were assessed for differential taxonomic and ARG profiling. Subsequently, Random Forest (RF)-based ML models were used to predict ARG profiles based on taxonomic composition and model validation on hospital wastewaters. Total ARG abundance was significantly higher in hospital wastewaters (15 ppm) than sewage (5 ppm), while the resistance towards methicillin, carbapenem, and fluoroquinolone were predominant. Although, Pseudomonas constituted major fraction, Streptomyces, Enterobacter, and Klebsiella were characteristic of hospital wastewaters. Prediction modeling showed that the relative abundance of pathogenic genera Escherichia, Vibrio, and Pseudomonas contributed most towards variations in total ARG count. Moreover, the model was able to identify host-specific patterns for contributing taxa and related ARGs with >90% accuracy in predicting the ARG subtype abundance. More than >80% accuracy was obtained for hospital wastewaters, demonstrating that the model can be validly extrapolated to different types of wastewater systems. Findings from the study showed that the ML approach could identify ARG profile based on bacterial composition including 16S rDNA amplicon data, and can serve as a viable alternative to metagenomic binning for identification of potential hosts of ARGs. Overall, this study demonstrates the promising application of ML techniques for predicting the spread of ARGs and provides guidance for early warning of ARBs emergence.

The present study was carried out to valorise cereal (rice and wheat) bran for the development of low-cost liquid consortium bioformulation. Different concentrations of bran-based liquid media formulations were evaluated for the growth of consortium biofertilizer cultures (Azotobacter chroococcum, Bacillus subtilis and Pseudomonas sp.). Among the bran-based formulations, wheat bran-based formulation WB5, exhibited the highest viable cell of 10.68 ± 0.09 Log₁₀ CFU/ml and 12.63 ± 0.04 Log₁₀ CFU/ml for Azotobacter chroococcum and Bacillus subtilis whereas for Pseudomonas sp., rice bran based bioformulation RB5 recorded maximum viability (12.71 ± 0.05 Log₁₀ CFU/ml) after 72 h of incubation. RB5¹ and WB5²liquid formulations were further optimized for enhanced shelf life using 5, 10 and 15 mM of trehalose, 0.05 and 0.1% carboxymethyl cellulose, and 0.5 and 1.0% glycerol. Following the peak growth at 72 h of incubation, a gradual decrease in the viable population of consortium biofertilizer cultures was observed in all the liquid formulations. The WB5 and RB5 formulations with 15 mM trehalose and 0.1% CMC, not only recorded significantly highest cell count of consortium biofertilizer cultures, but also maximally supported multi-functional traits i.e., phosphate and zinc solubilization, ammonia and IAA production up to 150 days. Further evaluation of seedling emergence and growth of wheat (PBW 826) under axenic conditions recorded WB5 amended with 15 mM trehalose-based consortium bioformulation to exhibit maximum emergence and growth of wheat seedlings. This low-cost liquid formulation can be used for large-scale biofertilizer production as a cost-effective liquid biofertilizer production technology.