Conventional Culture Methods Research Articles

Enhancing clinical microbiology for genomic surveillance of antimicrobial resistance implementation in Africa.

Surveillance is essential in the fight against antimicrobial resistance (AMR), to monitor the extent of resistance, inform prevention, control measures, and evaluate intervention progress. Traditional surveillance methods based on phenotypic antimicrobial susceptibility data offer important but limited insights into resistance mechanisms, transmission networks, and spread patterns of resistant bacterial strains. Fortunately, genomic technologies are increasingly accessible and can overcome these limitations. Genomics has the potential to advance traditional bacteriology in routine diagnosis and surveillance, it often relies on the initial isolation of bacterial strains from clinical specimens using conventional culture methods. Culture-based phenotypic characteristics are essential for making inferences about newly recognized genomic patterns. The Africa CDC Pathogen Genomics Initiative (Africa PGI) aims to enhance disease surveillance and public health partnerships through integrated, cross-continent laboratory networks equipped with the tools, human resource capacity and data infrastructure to fully leverage critical genomic sequencing technologies. For genomic surveillance of AMR, it is essential to optimize routine clinical microbiology laboratory services that are weak in many African countries. In this review, we outline shortcomings in clinical microbiology laboratories across Africa that compromise pathogen genomic epidemiology. We emphasize the necessity of investing in bacteriology and enhancing leadership capacity to fully capitalize on the advantages offered by genomic antimicrobial resistance (AMR) surveillance.

Improvement of Transplanting Rice Yield and Nitrogen Use Efficiency by Increasing Planting Density in Northeast China Under the Optimal Nitrogen Split-Fertilizer Applications

There are few studies on how nitrogen (N) fertilizer application rates and transplanting densities impact rice yield, root distribution, and N use efficiency in the cold regions of Northeast China. This research involved a two-year field trial utilizing Jinongda 667 as the material. In 2021, three N split-fertilizer applications—T1 (6:3:1), T2 (5:3:2), T3 (4:3:3)—and two transplanting densities—D1 (30 cm × 13.3 cm) and D2 (30 cm × 20 cm)—were compared with the conventional cultivation mode (T0: 175 kg N hm−2, 6:3:1), whereby the N application mode most suitable for increasing density was explored. In 2022, four N application levels—0 (N0), 125 (N1), 150 (N2), and 175 (N3) kg N hm−2—were assessed under the same density treatment to analyze the yield, resource utilization efficiency, and root traits of Jinongda 667. The results indicated that when the transplanting density was 30 cm × 13.3 cm, the application of 5:3:2 fertilizer was more conducive to improving rice yield. Increasing planting density under reduced N input significantly enhanced both rice yield and N use efficiency. In contrast to the conventional cultivation method (D2N3), the treatment of increased planting density (D1N2) under reduced N input led to a 21.2% rise in the number of panicles per square meter and an 8.6% boost in rice yield. Furthermore, increasing planting density under reduced N input significantly enhanced the agronomic efficiency of N fertilizer, the apparent utilization rate, and the N harvest index. It also boosted the SPAD value, photosynthetic rate, and the utilization efficiency of light and N resources in rice. However, it was noted that root enzyme activity decreased. This study demonstrated that increasing planting density, combined with the N application mode of 5:3:2 and an N application rate of 150 kg hm−2, maximized resource utilization efficiency, optimized root absorption capacity, and resulted in higher yields.

Advancements in Organoid Culture Technologies: Current Trends and Innovations.

Organoids have emerged as valuable tools in investigating disease mechanisms, drug efficacy, and personalized medicine due to their capacity to recapitulate crucial aspects of tissue physiology, including cell-cell interactions, heterogeneity, microenvironmental cues, and drug responses. Despite their broad applicability across various research domains, conventional organoid culture methods are plagued by several limitations that hinder research progress. These limitations include the inability to faithfully recreate tissue microenvironments, immune contexts, and vascular systems. Fortunately, ongoing advancements in organoid culture techniques are addressing these shortcomings. In this review, we provide a comprehensive overview of current mainstream organoid culture protocols. By evaluating these protocols, researchers can identify the most suitable experimental methods, thereby optimizing resource allocation and experimental outcomes.

High utility of bronchoalveolar lavage fluid metagenomic next-generation sequencing approach for etiological diagnosis of pneumonia

BackgroundFor patients with pneumonia, the rapid detection of pathogens is still a major global problem in clinical practice because traditional diagnostic techniques for infection are time-consuming and insensitive. Metagenomic next-generation sequencing (mNGS) is a novel technique that has the potential to improve pathogen diagnosis. This study aimed to investigate the microbiological diagnostic ability of mNGS compared with conventional culture and to determine the optimal time to test patients for pneumonia.MethodsA prospective study using data from June 2020 to June 2021 was performed at a tertiary teaching hospital in China. We included 56 patients from all adult patients with a clinical diagnosis of pneumonia. Blood and bronchoalveolar lavage fluid (BALF) samples were taken for simultaneous mNGS and conventional culture testing.ResultsAll 56 patients underwent both conventional culture and mNGS. Of these patients, 37 were diagnosed with severe pneumonia and 17 were diagnosed with non-severe pneumonia. The top three pathogenic bacteria detected by mNGS were Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Enterococcus faecium was detected more frequently in the non-severe pneumonia group (4 vs. 0, p < 0.05). The findings revealed that the detection rate of mNGS (84%) was superior to that of conventional culture methods (48%). Notably, the percentage of mNGS-positive BALF samples (46/56, 82.14%) was significantly greater than that of blood samples (27/56, 48.21%). The etiological comparison demonstrated that mNGS-positive samples, which received clinical approval, tended to be associated with a more normalized temperature, lower PCO2 levels, and a higher SOFA score than mNGS-negative samples (p = 0.022, p = 0.0.028, and p = 0.038, respectively).ConclusionsIn this study, we discovered that the etiology of lung infections frequently involves multiple pathogens. The use of mNGS in BALF is instrumental for detecting nonviral pathogens associated with lung infections. Although the rate of positive blood NGS results is significantly influenced by various clinical factors, for patients suspected of having viral, Legionella, or tsutsugamushi infections, plasma mNGS could serve as a complementary diagnostic tool.

Assessing the Impact of Climate Change on Methane Emissions from Rice Production Systems in Southern India

The impact of climate change on methane (CH4) emissions from rice production systems in the Coimbatore region (Tamil Nadu, India) was studied by leveraging field experiments across two main treatments and four sub-treatments in a split-plot design. Utilizing the closed-chamber method for gas collection and gas chromatography analysis, this study identified significant differences in CH4 emissions between conventional cultivation methods and the system of rice intensification (henceforth SRI). Over two growing seasons, conventional cultivation methods reported higher CH4 emissions (range: from 36.9 to 59.3 kg CH4 ha−1 season−1) compared with SRI (range: from 2.2 to 12.8 kg CH4 ha−1 season−1). Experimental data were subsequently used to guide parametrization and validation of the DeNitrification–DeComposition (DNDC) model. The validation of the model showed good agreement between the measured and modeled data, as denoted by the statistical tests performed, which included CRM (0.09), D-index (0.99), RMSE (7.16), EF (0.96), and R2 (0.92). The validated model was then used to develop future CH4 emissions projections under various shared socio-economic pathways (henceforth SSPs) for the mid- (2021–2050) and late (2051–2080) century. The analysis revealed a potential increase in CH4 emissions for the simulated scenarios, which was dependent on specific soil and irrigation management practices. Conventional cultivation produced the highest CH4 emissions, but it was shown that they could be reduced if the current practice was replaced by minimal flooding or through irrigation with alternating wetting and drying cycles. Emissions were predicted to rise until SSP 370, with a marginal increase in SSP 585 thereafter. The findings of this work underscored an urgency to develop climate-smart location-specific mitigation strategies focused on simultaneously improving current water and nutrient management practices. The use of methanotrophs to reduce CH4 production from rice systems should be considered in future work. This research also highlighted the critical interaction that exists between agricultural practices and climate change, and emphasized the need to implement adaptive crop management strategies that can sustain productivity and mitigate the environmental impacts of rice-based systems in southern India.

Unveiling the Human Brain on a Chip: An Odyssey to Reconstitute Neuronal Ensembles and Explore Plausible Applications in Neuroscience.

The brain is an incredibly complex structure that consists of millions of neural networks. In developmental and cellular neuroscience, probing the highly complex dynamics of the brain remains a challenge. Furthermore, deciphering how several cues can influence neuronal growth and its interactions with different brain cell types (such as astrocytes and microglia) is also a formidable task. Traditional in vitro macroscopic cell culture techniques offer simple and straightforward methods. However, they often fall short of providing insights into the complex phenomena of neuronal network formation and the relevant microenvironments. To circumvent the drawbacks of conventional cell culture methods, recent advancements in the development of microfluidic device-based microplatforms have emerged as promising alternatives. Microfluidic devices enable precise spatiotemporal control over compartmentalized cell cultures. This feature facilitates researchers in reconstituting the intricacies of the neuronal cytoarchitecture within a regulated environment. Therefore, in this review, we focus primarily on modeling neuronal development in a microfluidic device and the various strategies that researchers have adopted to mimic neurogenesis on a chip. Additionally, we have presented an overview of the application of brain-on-chip models for the recapitulation of the blood-brain barrier and neurodegenerative diseases, followed by subsequent high-throughput drug screening. These lab-on-a-chip technologies have tremendous potential to mimic the brain on a chip, providing valuable insights into fundamental brain processes. The brain-on-chip models will also serve as innovative platforms for developing novel neurotherapeutics to address several neurological disorders.

Unravelling the complexity of bovine milk microbiome: insights into mastitis through enterotyping using full-length 16S-metabarcoding

BackgroundMastitis, inflammation of the mammary gland, is a major disease of dairy cattle and the main cause for antimicrobial use. Although mainly caused by bacterial infections, the aetiological agent often remains unidentified by conventional microbiological culture methods. The aim of this study was to test whether shifts in the bovine mammary gland microbiota can result in initiation or progression of mastitis.MethodsOxford-Nanopore long-read sequencing was used to generate full-length 16S rRNA gene reads (16S-metabarcoding) to characterise the microbial population of milk from healthy and diseased udder of cows classified into five groups based on their mastitis history and parity.ResultsSamples were classified into six enterotypes, each characterised by a marker genus and several differentially-abundant genera. Two enterotypes were exclusively composed of clinical mastitis samples and displayed a marked dysbiosis, with a single pathogenic genus predominating and displacing the endogenous bacterial population. Other mastitis samples (all subclinical and half of the clinical) clustered with those from healthy animals into three enterotypes, probably reflecting intermediate states between health and disease. After an episode of clinical mastitis, clinical recovery and microbiome reconstitution do not always occur in parallel, indicating that the clinical definition of the udder health status does not consistently reflect the microbial profile.ConclusionsThese results show that mastitis is a dynamic process in which the udder microbiota constantly changes, highlighting the complexity of defining a unique microbiota profile indicative of mastitis.

Evaluation of physico-chemical quality and occurrence of Salmonella in water from food retail outlets

Salmonella is one among the most important biological contaminants in drinking water that possess safety hazards to human life. Faecal contamination of the water, indirectly from the household sewage discharge, municipal sewage etc. attributes to the repeated detection of Salmonella in water. This study aimed to investigate the occurrence of Salmonella and physico- chemical quality evaluation of samples of water collected from food retail outlets of Thrissur and Kollam districts. A total of 50 water samples (25 each from Thrissur and Kollam districts) used for cooking purpose were collected over a period of six months, March to August 2023. Water samples were subjected to comparative evaluation of physico-chemical qualities viz., pH, turbidity, total dissolved solids (TDS) and hardness. Collected water samples were examined for Salmonella spp. by conventional culture method and PCR. Genus specific and virulence genes of the isolates were detected using polymerase chain reaction. The occurrence of Salmonella spp. in samples of water collected from Thrissur was 24 per cent whereas in Kollam district it was only four per cent. The overall occurrence of Salmonella spp. in samples of water collected from both districts was 14 per cent. Physico-chemical parameters of water samples viz., pH, turbidity, total dissolved solids and hardness were within the BIS prescribed standards. The molecular characterisation of Salmonella spp. detected 16S rRNA in all isolates but invA and spvA genes were detected only in 14.28 per cent of the isolates. Hence proper disinfection of water and food sanitation in the retail outlets will help to minimize the risk of foodborne pathogens Keywords: Salmonella, water, physico-chemical quality, food retail outlets

Clinical Utility of a Multiplex PCR Panel (BioFire Joint Infection®) in the Adjustment of Empiric Antimicrobial Therapy: Experience in Pediatric Osteoarticular Infections.

This study aims to evaluate the impact of the PCR multiplex panel (BioFire JI®) on the diagnosis and management of pediatric osteoarticular infections. This retrospective study analyzed data from pediatric patients diagnosed with osteoarticular infections between January 2023 and April 2024. The effectiveness of the PCR multiplex panel in identifying pathogens was compared with traditional culture methods. In total, 50 patients were identified (66.6% male, 74% under 3 years of age). They were diagnosed as follows: septic arthritis in 46%, osteomyelitis in 26%, and septic osteoarthritis in 22%. An identifiable agent was isolated by conventional culture in 22 cases (44%). Kingella kingae was the predominant pathogen identified, accounting for 50% of cases (11/22), followed by Staphylococcus aureus (9/22). The BioFire JI® Panel PCR demonstrated a sensitivity of 93%, with a specificity of 63% when evaluated against synovial fluid culture as the reference standard. The panel identified seven additional pathogens not detected by conventional culture methods: 2/9 MSSA (22%), 1/1 S. pyogenes (100%), and 4/11 K. kingae (37%), increasing the yield by 14%. The rapid identification of pathogens facilitated timely and targeted therapeutic interventions. The PCR multiplex panel (BioFire JI®) improved the diagnosis of pediatric osteoarticular infections.

Efficient Treatment of Psoriasis Using Conditioned Media from Mesenchymal Stem Cell Spheroids Cultured to Produce Transforming Growth Factor-β1-Enriched Small-Sized Extracellular Vesicles.

Psoriasis is a common chronic inflammatory disease in which keratinocytes proliferate abnormally due to excessive immune action. Psoriasis can be associated with various comorbidities and has a significant impact on health-related quality of life. Although many systemic treatments, including biologic agents, have been developed, topical treatment remains the main option for psoriasis management. Consequently, there is an urgent need to develop topical treatments with minimal side effects and high efficacy. Mesenchymal stem cells (MSCs) exhibit excellent immune regulation, anti-inflammatory activities, and therapeutic effects, and MSC-derived extracellular vesicles (EVs) can serve as crucial mediators of functional transfer from MSCs. Therefore, this study aimed to develop a safe and easy-to-use emulsion cream for treating psoriasis using MSC conditioned media (CM) containing EVs. We developed an enhanced Wharton's jelly MSC (WJ-MSC) culture method through a three-dimensional (3D) culture containing exogenous transforming growth factor-β3. Using the 3D culture system, we obtained CM from WJ-MSCs, which yielded a higher EV production compared to that of conventional WJ-MSC culture methods, and investigated the effect of EV-enriched 3D-WJ-MSC-CM cream on psoriasis-related inflammation. Administration of the EV-enriched 3D-WJ-MSC-CM cream significantly reduced erythema, thickness, and scaling of skin lesions, alleviated imiquimod-induced psoriasiform lesions in mice, and ameliorated histopathological changes in mouse skin. The upregulated mRNA expression of inflammatory cytokines, including IL-17a, IL-22, IL-23, and IL-36, decreased in the lesions. In conclusion, we present here a new topical treatment for psoriasis using an MSC EV-enriched cream.

Genome sequencing analysis of the pncA, rpsA and panD genes responsible for pyrazinamide resistance of Mycobacterium tuberculosis from Indonesian isolates.

Developing the most suitable treatment against tuberculosis based on resistance profiles is imperative to effectively cure tuberculosis patients. Whole-genome sequencing is a molecular method that allows for the rapid and cost-effective detection of mutations in multiple genes associated with anti-tuberculosis drug resistance. This sequencing approach addresses the limitations of culture-based methods, which may not apply to certain anti-TB drugs, such as pyrazinamide, because of their specific culture medium requirements, potentially leading to biased resistance culture results. Thirty-four M. tuberculosis isolates were subcultured on a Lowenstein-Jensen medium. The genome of these bacteria was subsequently isolated using cetyltrimethylammonium bromide. Genome sequencing was performed with Novaseq Illumina 6000 (Illumina), and the data were analysed using the GenTB and Mykrobe applications. We also conducted a de novo analysis to compare the two methods and performed mutation analysis of other genes encoding pyrazinamide resistance, namely rpsA and panD. The results revealed mutations in the pncA gene, which were identified based on the databases accessed through GenTB and Mykrobe. Two discrepancies between the drug susceptibility testing and sequencing results may suggest potential instability in the drug susceptibility testing culture, specifically concerning PZA. Meanwhile, the results of the de novo analysis showed the same result of pncA mutation to the GenTB or Mykrobe; meanwhile, there were silent mutations in rpsA in several isolates and a point mutation; no mutations were found in the panD gene. However, the mutations in the genes encoding pyrazinamide require further and in-depth study to understand their relationship to the phenotypic profile. Compared to the conventional culture method, the whole-genome sequencing method has advantages in determining anti-tuberculosis resistance profiles, especially in reduced time and bias.

Environmental monitoring of Listeria monocytogenes contamination in dairy processing facilities combining culturing technique and molecular methods

Objectiveof the present study was to investigate Listeria contamination in Sardinian sheep cheese-making plants using culture and DNA-based methods. Food and environmental samples were collected from different surfaces of 14 facilities. Samples were collected using pre-moistened sponge swabs. Each site was sampled using two sponges used one for microbiological detection and the other for extraction of total DNA. The DNA was submitted to real time PCR targeting the prs gene for Listeria spp., the gene inlA for Listeria monocytogenes and the gene iap for Listeria innocua. DNA metabarcoding was also conducted on the total DNA. Overall were collected 254 environmental and 36 food samples. The prevalence of Listeria spp. and Listeria monocytogenes was 18.6% and 10.0%, respectively. Samples enrichment coupled with quantitative PCR (qPCR) showed a greater sensitivity than the conventional culture method with an overall prevalence of 12.1% for L. monocytogenes. Both, qPCR and ribosomal DNA-metabarcoding conducted on total DNA extracted from the sponges performed poorly in comparison to the culture-based method detecting the presence of Listeria genus in as little as 7 (2.4%) and 4 (1.4%) samples respectively. The present study supports the use of a culture enrichment coupled with qPCR for routine monitoring of contamination in food premises.

Epidemiology of pediatric meningitis and encephalitis in Japan: a cross-sectional study.

Information on the epidemiology and cause-specific clinical features of pediatric meningitis and encephalitis is limited, owing to conventional laboratory methods' limitations in identifying causative pathogens. The FilmArray Meningitis/Encephalitis (FA-M/E) panel, a molecular diagnostic tool, can detect 14 pathogens within 1 hour. We investigated meningitis and encephalitis epidemiology among children in Japan and FA-M/E panels' utility in their clinical management. This cross-sectional study was conducted among children aged 0-18 years admitted to seven regional hospitals in western Japan between October 2022 and September 2023. Cerebrospinal fluid (CSF) samples were collected from 221 children and tested using the FA-M/E panel. Clinical and microbiological data were reviewed retrospectively. Fifty-eight patients tested positive using the FA-M/E panel. Viral and bacterial pathogens were detected in 49 and nine cases, respectively. Human parechovirus and enterovirus occurred mainly in epidemic clusters during the summer and were primarily detected in young infants. Patients who tested positive for human parechovirus had a significantly higher frequency of sepsis-like manifestations and a lower frequency of CSF pleocytosis than did those who tested positive for enterovirus. CSF pleocytosis was absent in 30 patients who tested positive using the FA-M/E panel. Among the patients who tested positive for bacteria, three of the nine were not diagnosed using conventional culture methods, owing to prior antimicrobial therapy. The FA-M/E panel can identify bacterial and viral pathogens causing pediatric meningitis and characterize the epidemiology in local communities.IMPORTANCECulture and polymerase chain reactions have traditionally been used to identify microorganisms causing pediatric meningitis and encephalitis. However, the methods currently used to identify the causative microorganisms are limited, particularly in general hospitals. The FilmArray Meningitis/Encephalitis (FA-M/E) panel, a fully automated genetic testing system, can detect 14 pathogens using the multiplex polymerase chain reaction method. This study described the epidemiology of pediatric meningitis and encephalitis in Japan. The microorganisms causing acute meningitis and encephalitis in children in Japan were identified using the FilmArray Meningitis/Encephalitis panel. Testing cerebrospinal fluid using the FA-M/E panel is useful for the identification of the pathogen in children with community-acquired acute meningitis and encephalitis. This increases knowledge on the epidemiology and clinical manifestations of acute meningitis and encephalitis caused by specific pathogens and can be used to facilitate optimal patient management.

Augmented pathogen detection in brain abscess using metagenomic next-generation sequencing: a retrospective cohort study.

Brain abscess is a severe infection characterized by the accumulation of pus within the brain parenchyma. Accurate identification of the causative pathogens is crucial for effective treatment and improved patient outcomes. This 10-year retrospective, single-center study aimed to compare the detection performance of conventional culture methods and metagenomic next-generation sequencing (mNGS) in brain abscess. We reviewed 612 patients diagnosed with brain abscess and identified 174 cases with confirmed etiology. The median age was 52 years, with 69.5% males. Culture tests predominately identified gram-positive bacteria, particularly Streptococcus spp. Gram-negative bacteria, including Klebsiella spp., were also detected. However, mNGS revealed a more diverse pathogen spectrum, focusing on anaerobes (e.g., Fusobacterium spp., Parvimonas spp., Porphyromonas spp., Prevotella spp., and Tannerella spp.). mNGS exhibited significantly higher overall pathogen-positive rates in pus samples (85.0% vs 50.0%, P = 0.0181) and CSF samples (84.2% vs 7.9%, P < 0.0001) compared to culture. Furthermore, the detection rates for anaerobes displayed a notable disparity, with mNGS yielding significantly higher positive detections in both pus samples (50.0% vs 10%, P = 0.0058) and CSF samples (18.4% vs 0%, P = 0.0115) when compared to culture methods. The assistance of mNGS in pathogen detection, particularly anaerobes in brain abscess, was evident in our findings. mNGS demonstrated the ability to identify rare and fastidious pathogens, even in culture-negative cases. These results emphasize the clinical value of mNGS as a supplement for brain abscess, enabling more comprehensive and accurate pathogen identification.IMPORTANCEThe accurate identification of pathogens causing brain abscess is crucial for effective treatment and improved patient outcomes. In this 10-year retrospective study, the detection performance of conventional culture methods and metagenomic next-generation sequencing (mNGS) was compared. The study analyzed 612 patients with brain abscess and confirmed etiology in 174 cases. The results showed that culture tests predominantly identified gram-positive bacteria, while mNGS unveiled a broader diverse pathogen spectrum, particularly anaerobes. The mNGS method exhibited significantly higher overall rates of pathogen positivity both in pus and cerebrospinal fluid (CSF) samples, surpassing the culture methods. Notably, mNGS detected a significantly higher number of anaerobes in both pus and CSF samples compared to culture methods. These findings underscore the clinical value of mNGS as a supplement for brain abscess diagnosis, enabling more comprehensive and accurate pathogen identification, particularly for rare and fastidious pathogens that evade detection by conventional culture methods.

Role of Xpert PCR kit in assessing MRSA colonization in medical and surgical units of a tertiary care teaching hospital

Background and rationaleMethicillin resistant Staphylococcus aureus (MRSA) colonization increases the risk of MRSA infection. Detecting MRSA colonization can influence postoperative outcomes and prolong hospital stay. The conventional standard culture method for detecting MRSA colonization has limitations in terms of sensitivity and turnaround time. Hence, we sought out use of Xpert PCR kit for prompt evaluation of MRSA colonization to support MRSA prevention in a tertiary care hospital in Karachi, Pakistan.Materials and methodsDuring 1st April-31st December 2022, 290 nasal and skin swab samples were collected from 257 patients and processed using routine culture (as gold standard method) and PCR-based MRSA detection assay (MRSA Xpert).ResultsA total of two hundred and ninety (290) swab samples from 257 patients were obtained, 33 of which were paired. The overall prevalence of MRSA colonization was 12% by both methods, with 90% of cases classified as community-associated (CA-MRSA) whereas 10% as hospital-acquired (HA-MRSA). The colonized group showed a higher subsequent MRSA infection rate (11% vs. 3.5%) compared to the noncolonized group. Culture identified 11% of screening samples as MRSA positive, Xpert MRSA assay showed 100% sensitivity and 95% specificity. The cost of a single MRSA Xpert assay was $50 while MRSA culture cost around $7.50.ConclusionOur study findings suggest that the presence of MRSA colonization in our cohort of patients is consistent with the existing trends in hospital epidemiology. Both conventional culture and Xpert MRSA methods showed comparable efficacy for detection of MRSA colonization. Larger-scale studies are recommended to validate these findings conclusively.

Identification and Epidemiological Analysis of Antibiotic-Resistant Bacteria in the Oral Microbiome of the Population in Pakistan.

Background Antibiotic resistance in the oral microbiome poses serious health risks worldwide, particularly in developing countries like Pakistan. Public health efforts are challenged by the potential of the oral cavity to serve as a reservoir for resistant bacteria due to its frequent exposure to antibiotics. Objective This study aimed to identify and analyze the prevalence and epidemiology of antibiotic-resistant bacteria within the oral microbiome of the Pakistani population. Methodology A cross-sectional study was conducted at Akhtar Saeed Medical and Dental College, Lahore, and Gomal Medical College, Dera Ismail Khan, from January 2023 to December 2023. A total of 290 participants, aged 18 years or older, were recruited based on specific inclusion and exclusion criteria. Oral swabs were collected and analyzed using conventional culture methods. All descriptive and inferential statistical analyses were performed using SPSS version 25 (IBM Corp., Armonk, NY), with a significance level set at p <0.05. Results The most common antibiotic-resistant bacteria identified were Enterococcus faecalis (24.48%, n = 71), Staphylococcus aureus (27.24%, n = 79), and Streptococcus mutans (35.86%, n = 104). The most frequent resistances were to penicillin (32.14%, n = 93), tetracycline (23.45%, n = 68), and erythromycin (22.07%, n = 64). Recent antibiotic use was significantly associated with higher rates of resistance (p = 0.01), with 75.19% of individuals (n = 97) who had used antibiotics within the past three to six months showing resistance. Conclusion The study reveals a high prevalence of antibiotic-resistant bacteria, particularly to penicillin and tetracycline, in the oral microbiome of the Pakistani population.

Neglected Pulmonary Infection Caused by Exophiala dermatitidis Misidentified as Rhodotorula spp.

Exophiala dermatitidis is an emerging black fungus that causes pulmonary infections that may be underestimated by conventional culture methods. We encountered one case that initially appeared to be yeast and was misidentified as Rhodotorula spp. using a commercial identification kit. Thus, genetic identification and clinical background investigations were conducted on 46 strains of Rhodotorula spp. The sequences of the internal transcribed spacer and large-subunit RNA genes (D1/D2 regions) of 43 isolates, excluding two environmental isolates and one difficult-to-culture isolate, were determined and genetically identified. Notably, 22 isolates were identified as E. dermatitidis and misidentified as Rhodotorula spp. using the conventional method. Based on the exclusion criteria, the clinical information of 11 patients was retrospectively reviewed. Five cases (definite) had definite exacerbation of pulmonary infections due to E. dermatitidis, and six cases (possible) had undeniable infections. Of the 11 cases of pulmonary infection suggested to be caused by E. dermatitidis, comorbidities included two cases of chronic pulmonary aspergillosis (CPA), three cases of pulmonary non-tuberculous mycobacterial (NTM) infection and one case of pulmonary nocardiosis, suggesting a trend towards simultaneous detection of chronic pulmonary infections. Steroid and immunosuppressive drug use was observed in five cases, and β-D-glucan elevation was observed in three of five definite cases of pulmonary infections due to E. dermatitidis. The possibility of E. dermatitidis infection should be considered when Rhodotorula spp. are isolated from cultures of airway-derived specimens, and, in addition to CPA and NTM, identification of E. dermatitidis may be important in chronic pulmonary infections.

Intercropping salt-sensitive Solanum lycopersicum L. and salt-tolerant Arthrocaulon macrostachyum in salt-affected agricultural soil under open field conditions: Physiological, hormonal, metabolic and agronomic responses

Salinity is one of the important environmental risks affecting agricultural production in the world. Under this condition and with the conventional cultivation methods, glycophyte plants, like tomato, are subjected to many stresses, such as ion toxicity, osmotic stress, nutritional disturbance, oxidative damage and metabolic disorders, which cause growth inhibition and yield reduction. In this context, the main objective of our study was to compare the physiological, hormonal, metabolic and agronomic responses of tomato plants (Solanum lycopersicum L.) grown in monoculture (TM) or intercropping (TH) with the halophytic species Arthrocaulon macrostachyum in a salt affected soil. The results showed that the intercropping system (TH) reduced the soil electrical conductivity, and Na+ and Cl- contents, improving mineral nutrition in tomato plants compared to TM. In addition, TH decreased the osmotic stress, improved water potential and increased water use efficiency in tomato plants, whereas the integrity of gas exchange parameters were maintained; as a consequence, an increase in tomato yield was achieved. Moreover, the ratio of stress hormones (ABA, SA and JA) to growth regulating hormones (GA, auxins and cytokinins) decreased under TH. Metabolomic analysis showed clear defined patterns of differentially accumulated metabolites. Some of the metabolites with higher abundance in TH were linked to phenylpropanoid biosynthesis and phenylalanine metabolism, whereas alanine, aspartate and glutamate metabolism, monoterpenoid biosynthesis and butanoate metabolism pathways were downregulated. Our results support the importance of A. macrostachyum in the desalination of salt-affected soils and in the improvement of tomato yield in mixed culture. Indeed, this intercropping system offers farmers a low-cost biosolution that improves yields while respecting the environment.

Transient-resting culture after activation enhances the generation of CD8+ stem cell-like memory T cells from peripheral blood mononuclear cells

Adoptive cell therapy (ACT) has revolutionized the treatment of patients with cancer. The success of ACT depends largely on transferred T cell status, particularly their less-differentiated state with stem cell-like properties, which enhances ACT effectiveness. Stem cell-like memory T (TSCM) cells exhibit continuous self-renewal and multilineage differentiation similar to pluripotent stem cells. TSCM cells are promising candidates for cancer immunotherapies, whereas maintenance of a more stem-cell-like state before transfer is challenging. Here, we established a highly efficient protocol for generating CD8+ TSCM cells from peripheral blood mononuclear cells (PBMCs). The process involved activating PBMCs using anti-CD3 monoclonal antibody and RetroNectin, followed by a transient-resting culture period (24 h) and subsequent long-term expansion in vitro with interlukien-2. We report that this transient-resting culture after activation preserves CD8+ T cells in a stem memory phenotype (CD95+ CD45RA+ CCR7+) compared to the conventional culture method. Further, this approach reduces the expression of T cell immunoglobulin mucin-3, an exhaustion marker, and increases the expression of T cell factor-1, a master regulator of stemness even after long-term culture compared to the conventional culture method. In conclusion, our study presents a simplified and cost-effective method for generating and expanding CD8+ TSCM cells ex vivo. This approach streamlines the optimization of cancer immunotherapy using ACT.

Oscillation-static cycle cultivation enhances the synthesis of triterpenes and mycelium activity in Ganoderma lucidum

Ganoderma lucidum is a precious fungus with both edible and medicinal values and has a long history of medical use. Triterpenes as the main active components endow G. lucidum with anti-tumor, antioxidant, and other pharmacological activities. The present study endeavors to establish a proficient liquid-state fermentation technology for the enhanced production of triterpenes. In view of the limitations inherent in conventional submerged fermentation and oscillation-static two-stage cultivation, this study established an oscillation-static cycle cultivation process and optimized the cultivation conditions by building an artificial neural network model based on genetic algorithms. The cultivation conditions for the high-yield production of triterpenes were optimized as follows: 2.8 days of oscillation, 7.3 days of static cultivation, 0.2 day of oscillation, and 0.3 day of static cultivation. Under these conditions, the content of triterpenes reached 20.82 mg/g. The yield of triterpenes reached 129.09 mg/L, showing a remarkable increase of 324.78% compared with that of the Z10J0 method. Moreover, the established method shortened the cultivation cycle by 10.6 days. The mycelia cultivated under this regimen exhibited commendable anti-tumor and antioxidant activities. This study not only presents an economical liquid-state fermentation approach but also streamlines the fermentation flow, reduces fermentation duration, and effectively ameliorates drawbacks associated with conventional cultivation methods. In addition, this study gives valuable insights into the scaled application of liquid-state fermentation in the high-yield production of triterpenes, which showcases broad prospects.