Conventional Culture Methods Research Articles

A Comparative Study of Conventional and Automated Blood Culture System in Adult Patients

Introduction: Blood culture is considered as the gold standard for the diagnosis of bloodstream infection. Conventional blood culture system is less sensitive and takes longer duration for the detection of bloodstream infections whereas automated blood culture system is more sensitive and rapid in detecting causative organisms of bloodstream infections. This prospective study was undertaken to compare the automated blood culture system with the conventional blood culture system for the identification of microbial pathogens in bloodstream infections. Method: This prospective study was done in Department of Microbiology, Silchar Medical College & Hospital, Silchar for a period of 7 months from November 2021 to May 2022. Blood samples from the patients were inoculated into BD BACTEC Plus Aerobic culture vials for automated blood culture system and Brain Heart Infusion broth for conventional blood culture system. Positive bottles flagged by the automated machine were isolated and identified by doing routine subcultures on Blood agar and MacConkey agar and necessary biochemical tests. The conventional blood culture bottles were processed as per standard protocols. Result: Out of 123 samples, 21(17.09%) showed culture positivity by automated blood culture method and 15(12.19%) showed culture positivity by conventional blood culture method. The most common isolate was Staphylococcus aureus followed by Escherichia coli in automated method whereas the most common isolate in conventional method was Staphylococcus aureus followed by Klebsiella pneumoniae. Conclusion: This study concluded that automated blood culture method is more sensitive than conventional method and detects the presence of microorganisms rapidly causing bloodstream infections. Keywords: Bloodstream infection, Automated blood culture, Conventional blood culture

The effect of GdYVO4:Eu3+ nanocrystals on the intercellular adhesion of mesenchymal stem cells in vitro

Adult stem cells, such as MSCs, spontaneously differentiate in vitro. This makes it difficult both to study this important cell type and to grow large numbers of MSCs for clinical use. While conventional cell cultivation methods cannot cope with this problem, nanostructured materials science offers hope. The effect of small-sized spherical nanoparticles based on orthovanadates of rare-earth elements activated by europium (GdYVO4:Eu3+ nanoparticles, diameter 1–2 nm) on cell-cell adhesion of rat bone marrow mesenchymal stem cells (rBM-MSCs) in vitro was studied using electrophoretic separation of proteins, immunofluorescence and confocal laser scanning microscopy. Our study revealed that rBM-MSCs treated with small-sized GdYVO4:Eu3+ nanoparticles had a significant impairment of intercellular adhesion in vitro. The pre-incubation of mesenchymal stem cells of rat bone marrow with GdYVO4:Eu3+ nanocrystals at a non-toxic concentration of 0.5 µg/mL during 1 hour of cultivation did not lead to significant changes in cell monolayer, the number of cells and the area of cell bodies did not change. However, the density of the monolayer and the area of the cell field decreased after the incubation. The incubation of cells with nanoparticles led to an increase in the area of the intercellular gate – a location of disruption of cell adhesion, compared to cells without nanoparticles in culture medium. The pre-incubation of rBM-MSCs with nanocrystals caused no changes in the content of total cadherins in the plasma membrane; a decrease in the content of cytoplasmic calreticulin and an increase in the content of surface calreticulin; a decrease in the content of free calcium in the cytoplasm, and an increase in protein-bound intercellular calcium and calcium in the extracellular space. The colocalization analysis revealed that the colocalization of calreticulins with cadherins on the outer surface of the plasma membrane of cells significantly increased after the incubation with GdYVO4:Eu3+ nanocrystals. The paper proposes a possible mechanism of reducing the degree of adhesion by nanocrystals. This study emphasizes the possibility of modulating MSCs adhesion using GdYVO4:Eu3+ nanoparticles. The development of new technologies capable of mitigating adhesion is crucial for the development of regenerative strategies using stem cells.

Evaluating iQ-CheckTM real-time PCR to detect Salmonella from poultry environmental samples in Fraser Valley, British Columbia, Canada.

Salmonella is a ubiquitous pathogen that accounts for foodborne and livestock illnesses worldwide. Robust surveillance programs must be implemented to maintain human and animal health and limit economic losses. The poultry industry in particular demands the implementation of rapid Salmonella detection methods that will facilitate the timely availability of results in a manner allowing actions to be taken for the associated poultry products. One such method, the iQ-CheckTM real-time PCR, has significantly reduced turnaround times compared to conventional culture methods. In this study, a total 733 poultry environmental samples was received from farms in the Fraser Valley of British Columbia, Canada and the real-time PCR method was assessed for its ability to detect Salmonella in comparison to the currently used culture protocol. The iQ-Check real-time PCR method was effective at accurately screening out the majority of negative samples, and demonstrated a very strong correlation with the culture method. This was especially true when selective enrichment was performed before PCR, with sensitivity, specificity, and accuracy values reaching 100.0%, 98.5%, and 98.9%, respectively. These results demonstrate that rapid detection methods could be effectively introduced into current Salmonella surveillance workflows dealing with environmental poultry samples to reduce turnaround times and minimize economic impacts on producers.

Subaqueous 3D stem cell spheroid levitation culture using anti-gravity bioreactor based on sound wave superposition

BackgroundRecently, various studies have revealed that 3D cell spheroids have several advantages over 2D cells in stem cell culture. However, conventional 3D spheroid culture methods have some disadvantages and limitations such as time required for spheroid formation and complexity of the experimental process. Here, we used acoustic levitation as cell culture platform to overcome the limitation of conventional 3D culture methods.MethodsIn our anti-gravity bioreactor, continuous standing sonic waves created pressure field for 3D culture of human mesenchymal stem cells (hMSCs). hMSCs were trapped and aggerated in pressure field and consequently formed spheroids. The structure, viability, gene and protein expression of spheroids formed in the anti-gravity bioreactor were analyzed by electron microscope, immunostaining, polymerase chain reaction, and western blot. We injected hMSC spheroids fabricated by anti-gravity bioreactor into the mouse hindlimb ischemia model. Limb salvage was quantified to evaluate therapeutic efficacy of hMSC spheroids.ResultsThe acoustic levitation in anti-gravity bioreactor made spheroids faster and more compact compared to the conventional hanging drop method, which resulted in the upregulation of angiogenic paracrine factors of hMSCs, such as vascular endothelial growth factor and angiopoietin 2. Injected hMSCs spheroids cultured in the anti-gravity bioreactor exhibited improved therapeutic efficacy, including the degree of limb salvage, capillary formation, and attenuation of fibrosis and inflammation, for mouse hindlimb ischemia model compared to spheroids formed by the conventional hanging drop method.ConclusionOur stem cell culture system using acoustic levitation will be proposed as a new platform for the future 3D cell culture system.Graphical

Development of a Propidium Monoazide-Based Viability Quantitative PCR Assay for Red Sea Bream Iridovirus Detection

Red sea bream iridovirus (RSIV) is an important aquatic virus that causes high mortality in marine fish. RSIV infection mainly spreads through horizontal transmission via seawater, and its early detection could help prevent disease outbreaks. Although quantitative PCR (qPCR) is a sensitive and rapid method for detecting RSIV, it cannot differentiate between infectious and inactive viruses. Here, we aimed to develop a viability qPCR assay based on propidium monoazide (PMAxx), which is a photoactive dye that penetrates damaged viral particles and binds to viral DNA to prevent qPCR amplification, to distinguish between infectious and inactive viruses effectively. Our results demonstrated that PMAxx at 75 μM effectively inhibited the amplification of heat-inactivated RSIV in viability qPCR, allowing the discrimination of inactive and infectious RSIV. Furthermore, the PMAxx-based viability qPCR assay selectively detected the infectious RSIV in seawater more efficiently than the conventional qPCR and cell culture methods. The reported viability qPCR method will help prevent the overestimation of red sea bream iridoviral disease caused by RSIV. Furthermore, this non-invasive method will aid in establishing a disease prediction system and in epidemiological analysis using seawater.

Prediction of presence of fastidious bacteria by the Fully Automated Urine Particle Analyzer UF-1000i in the case of ineffective antimicrobial therapy for urinary tract infection

IntroductionRecent studies have reported associations between fastidious bacteria that are difficult to grow and isolate in conventional urine culture conditions and urinary tract infections (UTIs). Because the Fully Automated Urine Particle Analyzer UF-1000i (hereinafter referred to as “UF-1000i”) detects fastidious bacteria without being affected by culture conditions, owing to its flow cytometry-based principle, we evaluated the robustness of UF-1000i detection using clinical urine samples from patients with UTIs following ineffective antimicrobial therapy. MethodsA total of 150 patients diagnosed with UTIs were enrolled, and their laboratory findings were analyzed, focusing on the discrepancy in bacterial numbers between UF-1000i and conventional culture at each antimicrobial therapy effectiveness classification. In addition, gene identification was conducted by molecular analysis using 16S ribosomal RNA gene sequencing and next-generation sequencing (NGS) to elucidate the reason for the presence of fastidious bacteria in these samples. ResultsThe ineffective therapy cases showed more than 100-fold discrepancy in bacterial counts, with a higher proportion (30.8%) than effective therapy cases without secondary administration (5.7%) between the bacterial counts in UF-1000i and conventional culture methods. The presence rates of fastidious bacteria were 100% and 66.7% in discrepant cases of ineffective and effective without secondary administrations, respectively. ConclusionThis study suggests that discrepancies in bacterial numbers between the conventional culture method and UF-1000i measurement at the primary visit can predict the presence of fastidious bacteria, especially in cases of ineffective antimicrobial therapy.

Approaches to increase recovery of bacterial and fungal abortion agents in domestic ruminants.

Abortions in domestic ruminants cause significant economic losses to farmers. Determining the cause of an abortion is important for control efforts, but it can be challenging. All available diagnostic methods in the bacteriology laboratory should be employed in every case due to the many limiting factors (autolysis, lack of history, range of samples) that complicate the investigation process. The purpose of this study was to determine whether the recovery of diagnostically significant isolates from domestic ruminant abortion cases could be increased through the use of a combination of the existing aerobic culture andBrucellaselective method with methods that are commonly recommended in the literature reporting abortion investigations. These methods are examination of wet preparations and impression smears stained by the modified Ziehl-Neelsen method, anaerobic, microaerophilic,Leptospira, Mycoplasmaand fungal culture. Samples of placenta and aborted foetuses from 135 routine clinical abortion cases of cattle (n= 88), sheep (n= 25) and goats (n= 22) were analysed by the new combination of methods. In 46 cases, bacteria were identified as aetiological agents and in one case a fungus. Isolation ofBrucellaspecies increased to 7.4% over two years compared with the previous 10 years (7.3%), as well asCampylobacter jejuni(n= 2) andRhizopusspecies (n= 1).Salmonellaspecies (5.9%) andTrueperella pyogenes(4.4%) were also isolated more often. In conclusion, the approach was effective in removing test selection bias in the bacteriology laboratory. The importance of performing an in-depth study on the products of abortion by means of an extensive, combination of conventional culture methods was emphasised by increased isolation ofBrucella abortusand isolation ofC. jejuni. The combination of methods that yielded the most clinically relevant isolates was aerobic, microaerophilic,Brucellaand fungal cultures.

Bacterial and fungal profile of diabetic foot ulcer

Introduction: Polymicrobial biofilm in chronic diabetic foot wounds is a major challenge for the treatment and lead to several complications, including increased morbidity, mortality, and limb loss. Methodology: In this study, a spectrum of bacterial and fungal isolates was carried out in 66-foot ulcer samples from diabetic patients. All bacterial isolates were identified by conventional culture methods and biochemical identification tests. Filamentous fungi and yeasts were identified morphologically and by staining methods. Results: Among 66 samples, 62 (94%) samples were culture-positive, and four samples (6%) were culture-negative by the conventional culture method. Polymicrobial growth was found in 11 samples (17%) and monomicrobial in 51 wound samples (77%). Both Gram-positive and Gram-negative bacterial species were isolated. The number of Staphylococcus aureus isolates was more (61%). Fungi were isolated in 4 patients, and all the isolates were identified as Candida tropicalis. Several clinical complications were found including hypertension (47%), osteoarthritis (6%), rheumatoid arthritis (8%), cardiovascular disease (19%), peripheral vascular disease (20%), amputation (35%), foot cramps (71%) and edema (8%). Conclusion: Monomicrobial growth was predominant than polymicrobial growth, and S. aureus was the predominant pathogen isolated. Adequate treatment management of diabetic foot is needed to reduce the risk of amputation.

Three-Dimensional Cell Culture Methods in Infectious Diseases and Vaccine Research

Cells, the basic structures of all living organisms, reside in an extracellular matrix consisting of a complex three-dimensional architecture and interact with neighboring cells both mechanically and biochemically. Cell–cell and cell–extracellular matrix interactions form a three-dimensional network that maintains tissue specificity and homeostasis. Important biological processes in a cell cycle are regulated by principles organized by the microenvironment surrounding the cell. The conventional cell culture methods failed to mimic in vivo-like structural organization and are insufficient to examine features such as connectivity of cells, cellular morphology, viability, proliferation, differentiation, gene and protein expression, response to stimuli, and drug/vaccine metabolism. Three-dimensional cell culture studies are very important in terms of reducing the need for in vivo studies and creating an intermediate step. Three-dimensional cell culture methods have attracted attention in the literature in recent years, especially in examining the cellular distribution of organs in the presence of infectious diseases, elucidating the pathogenic mechanism of action of viruses, and examining virus–host interactions. This review highlights the use and importance of three-dimensional cell culture methods in the design and characterization of novel vaccine formulations and the pathogenesis of infectious diseases.

Water Use Efficiency and Economic Evaluation of the Hydroponic versus Conventional Cultivation Systems for Green Fodder Production in Saudi Arabia

The current study is aimed to assess water use efficiency and evaluate economic viability of hydroponic and conventional production of barley green fodder by keeping in view the water scarcity challenges in Saudi Arabia. A hydroponic system and open field experimental plot was used to evaluate the water use efficiency for different irrigation regimes. Economic indicators for both production systems are estimated and compared to accomplish economic assessment. Estimated indicators include returns from inputs and net profit; benefit-cost ratio; break-even levels of prices, production, and yield; returns over variable cost; and returns on investment. Results indicated that the yield of barley green fodder produced under hydroponic conditions overtopped the yield under conventional cultivation. Under hydroponic and conventional conditions, WUE was decreased with increasing the harvesting date. However, WUE for the hydroponic technique was much higher than the conventional one. The returns and net profits supported the conventional cultivation methods, where lower dry matter content coupled with higher fixed and variable costs incurred by the hydroponic technique outweighed returns leading to economic loss. Cost-benefit ratios, returns over investment, and break-even prices and yield suggested that growing barley fodder under the hydroponic technique is economically not suitable for small-scale farming. However, regarding water conservation, hydroponic barley cultivation showed superiority over conventional field cultivation. Further research on the adoption of hydroponic fodder cultivation is highly recommended for water-scarce arid regions, such as the Kingdom of Saudi Arabia.

Comparison of BACTEC versus conventional enrichment medium forperitoneal dialysate fluid culture.

Bacterial peritonitis remains a significant cause of mortality in patients on peritoneal dialysis (PD). Early detection of causative organisms and targeted antimicrobial treatment allow for better clinical outcomes. This study compares bacterial growth results from peritoneal dialysate in the BACTEC blood culture system vs. conventional culture. We conducted a prospective study on 46 patients with 63 consecutive episodes of suspected PD peritonitis between August 2020 and August 2021. PD dialysate was simultaneously sent to the laboratory in both BACTEC and sterile bottles. BACTEC bottles were incubated in the BD BACTEC FX system for 5 days. PD effluent transported from the sterile bottles was centrifuged at 3,000 rpm for 15 minutes; the supernatant was inoculated into cooked-meat broth for enrichment. Both incubation methods were extended to 14 days if microorganisms were seen on the Gram-stained smear. Recovery of isolated micro-organisms and time to detection (TTD) were compared. 26 episodes of suspected PD peritonitis based on clinical criteria were identified during the study period. The sensitivity of the BACTEC and the conventional culture methods was 50% and 42.3%, respectively (p=0.45). Seven samples had partial concordance or discordant results. McNemar's χ2-test revealed no statistical difference between either method (p=0.45). TTD was 18.9 ± 24.4 hours via the BACTEC method vs. 37 ± 16.5 hours in conventional cultures (p=0.014). The comparable sensitivities and similar yield in identifying pathogens could be due to the enrichment medium and prolonged incubation period. The shorter TTD for the BACTEC method could facilitate earlier confirmation of bacteriological diagnosis and subsequent treatment strategies.

Emergence of Unusual Microorganisms in Microflora of Pilonidal Sinuses: A Multiple Case Series.

Recent reports have noted an emergence of unusual organisms in microflora of pilonidal sinus (PNS); this study was undertaken to identify the primary microbial flora associated with infected primary PNS over a period of 1 year. A prospective multiple case series. A case series of 20 patients with primary PNS in a tertiary care center in Varanasi, India, was studied. The study was conducted at the Department of Microbiology and General Surgery, Institute of Medical Sciences, Varanasi, over a period of 1 year (September 2016 to July 2017). Purulent exudate (pus) samples were collected from 20 patients with primary PNS from the discharging sinuses by aseptic methods. Samples were assessed for aerobic and anaerobic flora by conventional culture and molecular methods. Antimicrobial susceptibility testing was done for bacterial isolates. Bacterial diversity was compared with the demographic and clinical profile of the sinuses by multiple correspondence analysis. Of the total cases, 11 (55%) had purulent discharge, among which all showed polymicrobial flora. The ratio of aerobic to anaerobic organisms was 1:2 (16/32). Escherichia coli (E. coli, 4, 36.36%) and Enterococcus faecalis (E. faecalis, 4, 36.36%) were commonly isolated. Bifidobacterium was the most frequent anaerobe. Detailed molecular analysis revealed the presence of Kocuria flava as an unusual pathogen. On statistical analysis, factors like male gender, increased body mass index, absence of hair in sinus, presence of features of hirsutism, and absence of Fusobacteria were closely associated with one another in these PNS cases. The case series revealed the predominance of anaerobes in primarily infected PNS cases. Bifidobacterium spp and unusual pathogens like K. flava were among the emerging pathogens in infected PNS. Use of better molecular diagnostic facilities in addition to the conventional methods might enhance the verified diversity of microorganisms in such cases.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients

Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

Development of Microphysiological Systems (MPSs) Based on Microfluidic Technology for Drug Discovery in Japan

Microphysiological systems (MPSs) based on microfluidic devices are attracting attention as an alternative cell assay platform to animal experiments in drug discovery. When we use microfluidic devices for cell culture, it is possible to experiment with various culture conditions that are difficult with conventional cell culture methods, such as fabrication of microstructures for cell placement, temporal and spatial control of liquid factors and adhesive conditions, and physical stimulation by flow and expansion/contraction. MPSs, which use microfluidic technology to construct the structure and function of physiological biological tissues and organs, are being commercialized and put to practical use worldwide with the entry of venture companies and pharmaceutical companies. Although research on the practical application of MPS in Japan has lagged far behind the efforts of Western countries, the Japan Agency for Medical Research and Development (AMED) launched the MPS Development and Research Project in FY2017 and established a system for MPS commercialization through industry-government-academia collaboration. The project is characterized by the formation of a consortium involving many researchers not only from academia but also from manufacturing and pharmaceutical companies with the aim of commercializing MPS devices. By FY2021, the final year of this project, several MPSs were successfully positioned in various stages of commercialization. This paper introduces two MPSs that the author was involved in commercializing in collaboration with domestic companies within the project.

Concordance between HB&L system and conventional culture method for bacterial antibiotic susceptibilities from positive blood samples at Hospital for Tropical Diseases from 03/2020 to 04/2021

Presently, shortening the time-to-result of antimicrobial susceptibility test (AST) is of the utmost importance for sepsis management in an effort to reduce mortality and antibiotic resistance rate. HB&L ((Human Biological Liquid Culture), a product of the Alifax company (Italy), can detect replicating microorganisms and their antibiotic resistance in a few hours with high sensitivity and specificity by using light scattering technique. This study aims to evaluate the correlation between rapid antibiogram using the HB&L system and the routine methods on positive blood cultures at the Hospital for Tropical Diseases in Ho Chi Minh City from March 2020 to April 2021. Gram-positive bacteria blood samples were tested against Cefoxitin, Vancomycin while Gram-negative bacteria samples were trialed with Ceftriaxone, Levofloxacin, Piperacillin-Tazobactam and Meropenem. The concordance of the two methods was defined by the categorical agreement (CA), very major errors (VME), major errors (ME) and minor errors (mE) in Cumitech 31A. Of the 81 blood culture samples containing Gram-negative bacteria, the CA of Ceftriaxone, Meropenem, Levofloxacin and Piperacillin-Tazobactam were 90%, 100%, 67% and 88%. The maximum VME was 22% for Levofloxacin. On the other hand, of the 32 Gram-positive samples, CA and VME of Cefoxitin were 53% and 65% respectively while CA of Vancomycin was 100%. The median time to receive rapid AST results of the HB&L system is 4.43 hours faster than the standard that.

Sığırlarda Mastitisin Teşhisinde Mikrobiyolojik Kültür ve Çiftlikte-Kültür Yöntemlerinin Karşılaştırılması

Bu çalışmada, bakteriyel nedenli inek mastitislerinin teşhisinde çiftlikte kültür ve konvansiyonel kültür yöntemlerinin karşılaştırılması, izole edilen bakterilerin bazı anibiyotiklere in vitro duyarlıklıklarının ortaya konulması amaçlandı. Çalışmada kanlı agar, MacConkey agar, Edwards medium ve Columbia CNA agar (CNA) kullanıldı. İn vitro antibiyotik duyarlılığının belirlenmesinde neomisin/basitrasin/tetrasiklin (30 μg/10 IU/30), sefapirin (30 μg), amoksisilin/klavulanik asit (2/1) (30 μg), penisilin (10 IU), tetrasiklin (30 μg), klindamisin (2 μg), kanamisin (30 μg) ve sefalotin (30 μg) diskleri kullanıldı. Konvansiyonal izolasyon ve identifikasyon yöntemlerinin uygulandığı çalışmada, örneklerin %90,3’ünden kültür pozitif sonuç alınırken, %9,7’sinde ise herhangi bir aerobik bakteri üremedi. Kanlı agarda yapılan izolasyonda kültür pozitif örneklerinin %17,9’unda (n: 14) Staphylococcus aureus, %61,6’sında (n: 48) Streptococcus spp., %7,7’sinde (n: 6) E. coli, %6,4’ünde (n: 5) Enterococcus spp., %3,8’inde (n: 3) maya ve %2,6’sında (n: 2) koagulaz negatif stafilokok (KNS) saptandı. MacConkey agara yapılan ekimlerin %60’ında (n: 6) E. coli, %30’unda (n: 3) Enterococcus spp. ve %10’unda (n: 1) maya belirlendi. Edwards mediuma ait ekimlerin %91,5’inde (n: 43) Streptococcus spp., %6,4’ünde (n: 3) Enterococcus spp., %2,1’inden (n: 1) maya ve KNS görüldü. CNA’a yapılan ekimlerin %20’sinden (n: 14) S. aureus, %68,6’sından (n: 48) Streptococcus spp., %4,3’ünden (n: 3) Enterococcus spp., %4,3’ünden (n: 3) maya ve %2,8’inden (n: 2) ise KNS izole edildi. Sonuç olarak, mastitisli inek sütlerinde bazı aerobik bakteriyel patojenlerin saptanmasına yönelik uygulanan çiftlikte kültür yönteminin, konvansiyonel kültür yöntemine benzer sonuçlar verdiği ve bu yöntemin enfeksiyona yönelik koruma ve kontrol programlarında kullanılabileceği kanısına varıldı.

Detection of Legionella pneumophila in the Water Samples of Food Industries and Hospitals in Bangladesh

Background: The human pathogen Legionella pneumophila causes a serious pneumonia-like respiratory disease called Legionnaires' disease, mainly in elderly and immunocompromised individuals. This pathogen can be found in the water distribution systems of large constructions with cooling towers which is a common phenomenon at present in Bangladesh due to its rapid economic growth. But there is a dearth of information on the incidence of Legionella in Bangladesh. Therefore, the current study aimed to investigate the presence of Legionella pneumophila in hospital and industrial water distribution systems in Dhaka, Bangladesh. Methods: A total of 114 water samples collected from two hospitals and five food industries were inoculated on the Legionella-specific medium Buffer Charcoal Yeast Extract (BCYE) agar medium before and after the treatment with acid, heat, or a combination of both. Samples producing Legionella-like colonies on BCYE agar medium were screened by Legionella Latex Test Kit, and the metagenomic DNAs obtained from these samples were analyzed by PCR using L. pneumophila-specific 16S rRNA primers. Results: Among 114 samples, Legionella-like colonies were observed in 30 water samples which demonstrated no agglutination in the Latex agglutination test. PCR analysis showed the presence of L. pneumophila in seven water samples, four in the potable water, chiller water, and cooling tower water of two different food industries, and three in ICU tap water, cooling tower water, and Fan Coil Units of two different hospitals. Sequence analysis of amplicons revealed that all seven sequences had 100% similarity with L. pneumophila. Conclusion: The presence of L. pneumophila in the water samples of local hospitals and food industries indicates that these habitats might serve as a potential site for Legionnaires' infection in Bangladesh. The results also showed that PCR, contrary to the conventional culture methods, could be more efficient and rapid in the identification of L. pneumophila.

Double transplantation as a climate resilient and sustainable resource management strategy for rice production in eastern Uttar Pradesh, north India

-Enhancing the productivity of rainfed crops, especially rice, while coping with climate adversities and saving critical natural resources is essential for ensuring the food and nutrition security of a growing population. With this context, the present study was undertaken to validate promising farm innovation and adaptation practices used by small-medium landholding farmers for rice cultivation in eastern Uttar Pradesh (UP), north India, as well as to examine the sustainability of innovative practices for large-scale adoption. For this, a 3-year study comprising extensive field surveys and experiments was undertaken to compare single transplantation (ST) and double transplantation (DT) in rice along with organic addition (farm-yard manure, FYM) on crop growth, yield, climate resilience, soil quality, and overall sustainability i.e., social (women involvements and labour productivity), environmental (water productivity and nutrient use efficiency), and economic (benefit:cost ratio) dimensions of sustainability. Field experiments were conducted in triplicate using two local rice varieties (MotiNP-360 and Sampurna Kaveri) in two agroclimatic zones, namely the middle Gangetic plains and the Vindhyan zone, in the Mirzapur district of eastern Uttar Pradesh. The DT practices of rice with and without farm yard manure (FYM) (replacing at a dose of 25% NPK) were evaluated over conventional methods of rice cultivation (i.e., ST, as control) and analysis was done periodically. The DT practice improved growth (p < 0.05), percent fertile tiller and grain (p < 0.05), and rice yield (15–20% higher than ST), while also improving soil quality, yield indices, water and labour productivity, and the benefit-cost ratio. The DT practice also resulted in early maturity (10–15 days earlier than ST), created more labour days for women, decreased lodging and pest/disease incidence, as well as a subsequent reduction in the use of synthetic chemical pesticides and associated environmental costs. Importantly, the residual effects of FYM application significantly improved (p < 0.05) the grain yield in subsequent years of cropping. Optimizing DT cultivation practices, preferably with FYM input for various agro-climatic regions, is essential for large-scale sustainable rice production under changing climatic conditions.

The application of targeted nanopore sequencing for the identification of pathogens and resistance genes in lower respiratory tract infections.

Lower respiratory tract infections (LRTIs) are one of the causes of mortality among infectious diseases. Microbial cultures commonly used in clinical practice are time-consuming, have poor sensitivity to unculturable and polymicrobial patterns, and are inadequate to guide timely and accurate antibiotic therapy. We investigated the feasibility of targeted nanopore sequencing (TNPseq) for the identification of pathogen and antimicrobial resistance (AMR) genes across suspected patients with LRTIs. TNPseq is a novel approach, which was improved based on nanopore sequencing for the identification of bacterial and fungal infections of clinical relevance. This prospective study recruited 146 patients suspected of having LRTIs and with a median age of 61 years. The potential pathogens in these patients were detected by both TNPseq and the traditional culture workups. We compared the performance between the two methods among 146 LRTIs-related specimens. AMR genes were also detected by TNPseq to prompt the proper utilization of antibiotics. At least one pathogen was detected in 133 (91.1%) samples by TNPseq, but only 37 (25.3%) samples contained positive isolates among 146 cultured specimens. TNPseq possessed higher sensitivity than the conventional culture method (91.1 vs. 25.3%, P < 0.001) in identifying pathogens. It detected more samples with bacterial infections (P < 0.001) and mixed infections (P < 0.001) compared with the clinical culture tests. The most frequent AMR gene identified by TNPseq was bla TEM (n = 29), followed by bla SHV (n = 4), bla KPC (n = 2), bla CTX-M (n = 2), and mecA (n = 2). Furthermore, TNPseq discovered five possible multi-drug resistance specimens. TNPseq is efficient to identify pathogens early, thus assisting physicians to conduct timely and precise treatment for patients with suspected LRTIs.

Multiplex high-resolution melting assay for simultaneous detection of five key bacterial pathogens in urinary tract infections: A pilot study.

The diagnosis of urinary tract infections (UTIs) is usually based on the results of urine culture, but it is time-consuming, labor-intensive and has a low sensitivity. The aim of this study was to develop multiplex high-resolution melting assay (MHRM) for the simultaneous detection of five common bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Staphylococcus saprophyticus, Enterococcus faecalis, and group B streptococci (GBS)) directly from urine samples. A total of 287 urine specimens were evaluated by HRM assay and the results were compared with the conventional culture method. Five different melt curves generated and differentiated five bacterial pathogens. The detection limit of the MHRM assay was 1.5 × 103 CFU/ml for E. coli and K. pneumoniae and 1.5 × 102 CFU/ml for S. saprophyticus, E. faecalis and GBS. Compared to culture, the specificity of the MHRM assay ranged from 99.3 to 100%, and sensitivity 100% for all test pathogens. The MHRM assay developed in the current study might be functional tool for the diagnosis of UTIs and has the potential for direct detection of the organism in the clinical samples. Additionally, it creates results in less than 5 h, helping clinicians to start treatment with appropriate antimicrobial agents. This method could be a useful supplement to urine culture.