Journal of Advances in Microbiology

Microbial, Plant and Animal Based Strategies for Remediation of Heavy Metals: A Review

2026-05-23T13:29:00+00:00

Heavy metal contamination is a major environmental concern caused by industrialization, mining, agriculture, and urban activities. Toxic metals such as cadmium (Cd), lead (Pb), mercury (Hg), chromium (Cr), arsenic (As), and nickel (Ni) are persistent, non-biodegradable, and harmful to ecosystems and human health. Conventional remediation techniques are often expensive and may generate secondary pollution, prompting the need for sustainable alternatives. This review highlights microbial, plant, and animal-based strategies for heavy metal remediation. Microbial approaches employ bacteria, fungi, and algae that remove metals through biosorption, bioaccumulation, biotransformation, and redox processes. Phytoremediation uses plants, particularly hyperaccumulators, to extract, stabilize, or filter metals from contaminated soils and water. Animal-based methods, such as vermiremediation and aquatic bioindicators, also contribute to metal sequestration and ecological restoration. Integrated systems, including microbe-assisted phytoremediation and constructed wetlands, further enhance remediation efficiency.

Substrate Pretreatment for Efficient Bio-ethanol Production: A Review

2026-05-27T09:22:59+00:00

Fermentable sugars are crucial in bioethanol production, as they serve as the primary substrate for microbial fermentation, while microorganisms like yeast convert these sugars into ethanol and other byproducts like carbon dioxide. There is a need to break down complex carbohydrates into fermentable sugars, the process begins with pretreatment. Pretreatment is a crucial step in bioethanol production, particularly when using starchy materials and lignocellulosic biomass. Substrate is prepared by such pretreatment for efficient enzymatic hydrolysis and subsequent fermentation. The primary goal of pretreatment is to disrupt the complex structure of starch, cellulose, hemicellulose and lignocellulosic materials, making them more accessible to enzymes that break them down into fermentable sugars. This is achieved by methods that remove lignin, increase surface area, and modify the crystallinity of cellulose. Therefore, the aim of this research was to review substrate and its treatment for efficient bio-ethanol production by microorganisms. Fermentable feedstock, starchy substrate, saccharification of starch with enzymes such as alpha-amylase, beta-amylase and gamma-amylase were reviewed. Moreover, hemicellulose substrates, lignocellulosic biomass and chemical pretreatment of lignocellulosic substrates, such as acid hydrolysis and alkaline hydrolysis with the formation of fermentation inhibitors during hydrolysis, were discussed. Mechanical pretreatment of lignocellulosic substrate using irradiation, thermal energy, gases, and finally biological pretreatment using lignolytic fungi were mentioned. In conclusion, for optimal bioethanol production, there is a need for substrate pretreatment, especially where complex carbohydrates are involved, but the method should be exploited to avoid production of inhibitors that could negatively impact enzymatic hydrolysis or fermentation process.

National Landscape of Antimicrobial Resistance in the Philippines: A Systematic Review of Surveillance and Clinical Data

2026-06-01T10:30:59+00:00

Background: Antimicrobial resistance (AMR) is an escalating global public health threat that reduces the effectiveness of antimicrobial therapies and contributes to increased morbidity, mortality, and healthcare costs. In the Philippines, rising antibiotic consumption, self-medication, and inconsistent antimicrobial stewardship practices have accelerated the emergence of resistant pathogens. However, existing evidence remains fragmented across surveillance reports and clinical studies, limiting a comprehensive understanding of national resistance patterns.

Methods: This study employed a systematic review design in accordance with PRISMA 2020 guidelines and was registered in the PROSPERO database (CRD420261360657). A comprehensive literature search was conducted using PubMed/MEDLINE, Scopus, and Web of Science, supplemented by national surveillance reports. Studies published from January 2008 to April 2026 reporting antimicrobial resistance data from human clinical isolates in the Philippines were included. Eligible studies were screened based on predefined criteria, and data were extracted and synthesized using a narrative descriptive approach.

Results: A total of 14 studies met the inclusion criteria. Findings consistently identified Gram-negative pathogens, particularly Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, as the primary contributors to antimicrobial resistance. High resistance rates to critical antibiotics, including carbapenems and third-generation cephalosporins, were observed, with Acinetobacter baumannii demonstrating resistance levels of up to 56%. Increasing resistance trends were noted following the COVID-19 pandemic, alongside strong associations between antimicrobial consumption and resistance patterns. Molecular evidence revealed the role of clonal dissemination and horizontal gene transfer, with key resistance genes such as blaNDM (gene encoding New Delhi metallo-β-lactamase, an enzyme that confers resistance to carbapenem antibiotics) and blaOXA (gene encoding OXA-type β-lactamase enzymes, which confer resistance to β-lactam antibiotics, including carbapenems in some variants) identified. AMR was associated with substantial clinical burden, including mortality rates of up to 60% in severe infections.

Conclusion: Antimicrobial resistance in the Philippines is a growing and significant public health concern predominantly driven by multidrug-resistant Gram-negative pathogens. Strengthening antimicrobial stewardship, enhancing surveillance systems, and reinforcing infection prevention and control measures are essential to mitigate the increasing burden of AMR.

Vancomycin-Resistant Enterococcal Infections: A Report of Three Clinical Cases and Literature Review

2026-06-03T10:40:16+00:00

Introduction: Enterococci are Gram-positive bacteria that are part of the normal human gut flora but have emerged as major nosocomial pathogens, causing severe infections such as bacteremia, urinary tract infections, and peritonitis. Enterococcus faecium is particularly concerning due to its remarkable ability to acquire resistance genes, especially vanA and vanB, leading to vancomycin resistance.

Materials and Methods: Three cases of vancomycin-resistant Enterococcus faecium (VRE) infection were identified in the onco-hematology, pediatrics, and urology departments. Bacterial identification and susceptibility testing were performed using the API gallery and E-test, complemented by the BD Phoenix™ automated system, while the presence of vanA and vanB genes was confirmed by multiplex PCR.

Clinical Cases: All patients had risk factors for VRE infection, including prior antibiotic exposure, invasive devices, or immunosuppression. The E. faecium isolates were multidrug-resistant, showing susceptibility only to linezolid. Clinical outcomes were favorable following targeted antibiotic therapy and, when indicated, removal of the infected device.

Discussion: Vancomycin-resistant enterococci, especially E. faecium, are an increasing global health concern due to multidrug resistance and healthcare-associated infections. VRE prevalence has significantly increased worldwide, reaching 32.5% in Europe in 2023 and 73.5% of healthcare-associated E. faecium infections in the United States. Their spread is mainly linked to the vanA and vanB resistance genes and to biofilm formation, which enhances persistence, colonization, and antibiotic resistance in hospital settings.

Conclusion: Rapid identification of VRE and close collaboration between microbiology laboratories and infection control teams are crucial to prevent nosocomial transmission. These findings highlight the importance of continuous surveillance and enhanced awareness of VRE infections within healthcare facilities.

Studies on Standardization of Activation Process of Persulfate for Decontamination against Food Pathogens

2026-05-20T11:54:44+00:00

The present investigation was carried out to standardize the activation process of sodium persulfate for its antimicrobial application against selected foodborne pathogens, namely Escherichia coli, Listeria monocytogenes and Salmonella typhimurium. In this study, sodium persulfate (PS) was activated using three approaches, viz., ferrous sulphate (FeSO₄), sodium hydroxide (NaOH) and ultraviolet-C (UV-C) radiation. Different concentrations of PS (30-90 mmol/L) and exposure times (1-5 min.) were tested to evaluate their efficiency. All activation methods produced a significant reduction in microbial counts (p < 0.0001), with inhibition generally increasing with concentration and treatment time. Complete inactivation was achieved in several cases, such as FeSO₄ activation at 50 mmol/L for 1 min. against E. coli and L. monocytogenes, and NaOH activation at 90 mmol/L for 3 min. against L. monocytogenes. In the case of S. typhimurium, complete inhibition was obtained at 30-40 mmol/L PS activated with FeSO₄ or NaOH. Although UV-C activation was effective, it required longer exposure times (45-60 min.) which limit its practical use. From the study, three optimized treatments were identified: PS activated with FeSO₄ (50 mmol/L, 1 min.), PS activated with NaOH (50 mmol/L, 1 min.) and PS activated with NaOH (90 mmol/L, 3 min.). The results suggest that activated sodium persulfate could be considered a potential alternative to conventional chlorine-based sanitizers for microbial decontamination in fresh produce handling.

Exploring the Fermentation Potential of Indigenous Yeasts from Amelie Mango Waste of Côte d’Ivoire: Technological and Functional Property

2026-05-22T08:01:14+00:00

Mango plays an important role in the economic development of production areas. Mango trees are cultivated in many subtropical and tropical countries, including Côte d'Ivoire. This study aimed to valorize the waste from the 'Amélie' variety mango. To do so, yeasts were isolated from the mango pulp and screened to select potential starters. Thus, microbiological analyses made it possible to isolate and identify twenty-six (26) yeast isolates grouped into 2 based on colony and cell morphology. Among the 26 (twenty-six) strains, 19.23% had strong catalase activity; 23.07% had medium activity, and 57.69% had weak activity. The majority of isolates (61.53%) did not produce acetic acid, while a minority (38.46%) did. Only 19.23% of the isolates are capable of producing proteases, and 7 have high fermentative power. These isolates showed different behaviors depending on the variation of the culture medium's pH and the incubation temperature. Moreover, mangoes of the Amelie variety in a state of rot contain yeast isolates of interest for biotechnological applications, particularly for the production of alcoholic beverages such as wine, etc.

Effect of Temperature, pH and Relative Humidity on Growth of Root Rot Disease of Soybean

2026-05-25T09:37:51+00:00

Root rot of soybean constitutes a widespread and serious disease constraint in Maharashtra and several other soybean-growing regions of India, leading to substantial yield reductions. The incidence and severity of the disease are often exacerbated under conditions of high temperature and low soil moisture, as well as other unfavourable environmental stresses. Under such conditions, plants become physiologically weakened, thereby increasing their susceptibility to pathogen infection and disease development. Rhizoctonia bataticola causal agent of root rot and charcoal rot in various pulse crops. Due to fluctuation of temperature and climatic disturbance in the arid region is quite difficult to manage the root rot. Due to favourable weather circumstances, the disease spread widely across the state and resulting in significant yield losses. To reduce the number of sclerotia in soil or to minimise the contact of the inoculum and the host, six different temperatures and four pH levels were tested for their suitability on mycelial growth and sclerotial formation of the Rhizoctonia bataticola in vitro conditions. Maximum mycelial growth (88.33 mm) was observed at 30 ^oC temperature followed by 25ºC (66.33 mm), 35ºC (64.00 mm), 20 ºC (54.33) and 40ºC (27.00 mm) and maximum dry mycelial diameter (86.25 mm) was observed at 6.5 pH followed by pH level 5.5, 7.5, and 8.5 with colony diameter 71.33, 55.58, mm and 47.00 mm of the R. bataticola respectively. Root rot of soybean constitutes a widespread and serious disease constraint in Maharashtra and several other soybean-growing regions of India, leading to substantial yield reductions. The incidence and severity of the disease are often exacerbated under conditions of high temperature and low soil moisture, as well as other unfavourable environmental stresses. Under such conditions, plants become physiologically weakened, thereby increasing their susceptibility to pathogen infection and disease development.

Prevalence of Methicillin-resistant Staphylococcus aureus and Co-existence of Diverse Resistance Profiles in Pastry Products Sold in the City of Abidjan (Côte d'Ivoire)

2026-05-25T12:17:43+00:00

In the city of Abidjan, consumption of pastry products is common. However, these foodstuffs could be transmission channels of resistant bacteria, including methicillin-resistant Staphylococcus aureus, and lead to public health concerns. The current study, realized from 2^ndMarch to April 15^th2021, aims at assessing prevalence of methicillin-resistant S. aureus in pastry products sold in Abidjan along with analyzing other resistance profiles associated with these strains. Susceptibility testing for antibiotics commonly prescribed in the management of infections caused by this pathogen was performed on 578 S. aureus isolates from crescents (247), chocolate bread (207) and Creamed cakes (124), using the Mueller Hinton agar diffusion method. The mecA gene, responsible for methicillin resistance, was amplified and detected by PCR. Results revealed complete (100%) resistance of strains to oxacillin and cefoxitin, as well as high rates of resistance to levofloxacin (87.5%) and clindamycin (66.6%). The resistance profiles observed to Kanamycin and Tobramycin (KT) was 13.8%, the constitutive resistance to Macrolide-Lincosamin-Streptogramin (MLSb cons) was 11.8% and the high-level resistance to Kanamycin, Tobramycin, and Gentamicin KTG (7.6%). Overall rate of multi-resistance was 36.1%, with 18.3% for crescents, 11.9% for chocolate bread and 5.9% for creamed cakes. Finally, 24.9% of isolates resistant to methicillin, were confirmed by presence of \(\beta\)-Lactams resistance coding gene cassette (mecA). The highest contamination of foodstuffs was observed in the vicinity of Bus Stations and Markets (11.2%). Multidrug resistance represents a crucial public health concern, highlighting as a result, urgency of increased surveillance and implementation of good hygiene practices.

Antibacterial Activity of Different Solvent Leaf Extracts of Tithonia diversifolia (Hemsl.) A. Gray (Asteraceae): An Invasive Alien Medicinal Plant in South Africa

2026-05-26T11:58:13+00:00

Background and Aims: Antimicrobial resistance (AMR) has emerged as a major global public health concern, significantly reducing the efficacy of conventional antibiotics and complicating the treatment of infectious diseases. The rapid emergence and dissemination of resistant microbial strains have intensified the search for novel antimicrobial agents from natural sources. Medicinal plants have continued to attract considerable scientific interest owing to their rich repository of bioactive secondary metabolites. Tithonia diversifolia (Hemsl.) A. Gray, an invasive plant species widely distributed across Africa, is extensively employed in traditional medicine for the treatment of various ailments, suggesting the presence of pharmacologically active compounds. This study evaluated the in vitro antibacterial activity of different solvent leaf extracts of T. diversifolia using solvents of varying polarity.

Study Design: To evaluate the in vitro antibacterial activity of T. diversifolia leaves using solvents of varying polarity.

Place and Duration of Study: Fresh plant materials of T. diversifolia were collected from wild growing plants along Main Road, Vulindlela, off University of Zululand, KwaDlangezwa, KwaZulu-Natal, South Africa.

Methodology: Sequential extraction of powdered air-dried leaves of T. diversifolia was carried out using n-hexane, chloroform, ethyl acetate, and methanol. Antibacterial activity of the extracts was evaluated against selected Gram-positive and Gram-negative bacterial strains using agar disc diffusion and broth microdilution techniques. Ciprofloxacin and gentamycin were used as standard reference antibiotics. Zones of inhibition (IZ) and minimum inhibitory concentrations (MICs) were determined for all extracts.

Results: All solvent extracts demonstrated varying degrees of antibacterial activity against the tested bacterial strains. The ethyl acetate extract exhibited the strongest antibacterial activity, producing inhibition zones ranging from 10.03 ± 0.3 to 20.70 ± 0.8 mm and MIC values between 0.6 and 5.0 µg/mL. The hexane extract also showed pronounced antibacterial effects with comparable inhibition zones and MIC ranges. In contrast, methanol and chloroform extracts exhibited moderate to weak antibacterial activities. Overall, Gram-positive bacteria were more susceptible to the extracts than Gram-negative bacteria, possibly due to structural differences in their cell walls. The superior activity observed in the ethyl acetate fraction suggests that moderately polar solvents were more effective in extracting bioactive antibacterial phytoconstituents from T. diversifolia. These findings are consistent with previous reports indicating that solvent polarity significantly influences phytochemical recovery and biological activity.

Conclusion: The findings of this study demonstrate that different solvent leaf extracts of Tithonia diversifolia possess significant in vitro antibacterial activity, particularly the ethyl acetate and hexane fractions. The enhanced activity observed in moderately polar solvent extracts suggests the presence of potent bioactive phytochemicals with broad-spectrum antibacterial potential. These results support the ethnomedicinal use of T. diversifolia and highlight its potential as a promising source of novel antibacterial agents. Further phytochemical isolation, purification, and characterization studies are recommended to identify the active compounds responsible for the observed antibacterial effects and to explore their potential application in antimicrobial drug development.

Cholera Epidemiology, Surveillance and Response in Cameroon (1960-2010)

2026-05-27T12:01:17+00:00

Background: The burden, persistent and increasing number ofcholera epidemics in Cameroon has been a major concern. To strengthen cholera surveillance, Cameroon in partnership with international health corporations adapted the integrated disease surveillance and response strategy. This article describes cholera outbreaks during 1960-2010. Also, it discusses the evolution of health policies, surveillance and the deployment of means for the fight against cholera in Cameroon.

Methods: Semi-structured questionnaires were administered to resource persons Cameroon. Written sources which included archives, books, articles, thesis, dissertation and internship reports were also consulted.

Results: Cameroon faced a huge cholera burden from 1960 to 2010 with the annual number of reported cases and deaths fluctuating with 24495 cases and 1887 deaths recorded between 1971 and 1980, 6158 cases and 1028 deaths recorded between 1981 and 1990, 12906 cases and 2476 deaths between 1991 and 2000 and 17927 cases and 1044 deaths between 2001 and 2010. The cholera burden fell progressively from 2010 to 202 with just four outbreaks recorded from 2018 to 2023 including 1,628 cases in 2018, 360 cases in 2019, 1,889 cases in 2020 cases and over 13.000 cases in 2023. Cholera data was usually collected in the peripheral level and forwarded at the Regional and finally to the Ministry of Public Health.

Conclusion: Prolonged and recurrent cholera outbreaks have been observed in Cameroon and have increased in intensity and geographic despite measures put in place by the Cameroon government and its partners. Strengthening surveillance and improving water sanitation schemes, and sensitization would be key in addressing cholera epidemics.

Shelf-life Extension of Nigerian Traditional Bean (Vigna unguiculata) Pudding (Moinmoin) by HACCP-guided Interventions in the Preparation Process

2026-05-30T12:42:26+00:00

Background: Moinmoin is an inexpensive popular Nigerian traditional break-fast bean-pudding, which deteriorates within 24 hours of preparation due to microbial activities. The popularity is an impetus for seeking ways of extending the shelf-life.

Aim: The potential of extending moinmoin shelf-life using HACCP-guided control interventions in the cooking process was therefore, investigated.

Place and Duration of Study: This was undertaken in Delta State University, Abraka, Nigeria, over a period of 6 months.

Study Design and Methodology: Samples were collected from 3 roadside eateries at each step of the preparation process (soaking water, bean paste, ingredients and swabs from blender, cooking pots and packaging materials) and analysed for bacteria population using Nutrient and MacConkey agar. Isolates were identified by morphological and biochemical tests while hygienic practices were assessed with structured questionnaire. Control interventions were by pasteurization, blanching of ingredients and disinfection of equipment.

Results: Population of bacteria isolates varied with moinmoin preparation steps (2.5-6.4 log cfu/g/cm²/ml) with E. coli, Staphylococcus, Bacillus, Pseudomonas, Klebsiella, Salmonella, Enterobacter and Proteus as the constituents. Intervention measures eliminated these bacteria except Bacillus with low counts (4 cfu/g) in the bean paste. All the bacteria grew better in the bean paste than in the RTE moinmoin, which contained onion and pepper (P=0.000-0.001). Hygienic handling was poor (<50% of max. score). The addition of onion and pepper to the paste tended to retard the growth of the pasteurization-injured Bacillus sp. in shelfed RTE moinmoin for 72 h. Respondents (>50%) indication of dissatisfaction with taste, colour and aroma of shelved RTE which came after 72 h corroborated the 72-h shelf-stability.

Conclusion: The HACCP-guided intervention measures eliminated all other bacteria and retarded growth of Bacillus, the only surviving deteriogen, and consequently extended shelf-life of RTE moinmoin from <24 h to 72 h.

The Epidemiology of Cholera, Measles, Cerebrospinal Meningitis and Poliomyelitis in Cameroon 1960-2010

2026-05-30T12:47:59+00:00

Background: Since its independence, Cameroon has been faced with an upsurge in epidemics. These include epidemics such as cholera, poliomyelitis, measles and cerebrospinal meningitis which have marked the course of history in Cameroon, with adverse demographic, political, economic and social consequences. Therefore, we aimed to show resurgence of epidemics, and to highlight the difficulties and structural inadequacies of the Cameroonian health system.

Methodology: Data were obtained through review of archives from the Ministry of Health, and semi-structured questionnaires, interviews administered to resource persons at the Regional Delegation of Public Health and some NGO’s in Centre, Adamawa, North, Far north, littoral and west Regions of Cameroon.

Results: The reported number of annual cases and deaths from epidemics observed have been fluctuating. For measles: between 1971 and 1980 (45522 cases and 3317 deaths), 1981 to 1990, (30744 cases and 4209 deaths), 1991 to 2000 (85826 cases and 3188 deaths) and 2001 to 2010 (32489 cases and 4178 deaths). For cholera: between 1971 and 1980 (24495 cases and 1887 deaths), 1981 to 1990, (6432 cases and 1028 deaths), 1991 to 2000 (12906 cases and 2476 deaths) and from 2001 to 2010 (17926 cases and 1044 deaths). For cerebrospinal meningitis: 1971 to 1980 (12082 cases and 5319 deaths), 1981 to 1990, (24811 cases and 7780 deaths), 1991 to 2000 (34245 cases and 7390 deaths) and from 2001 to 2010 (1483 cases and 278 deaths). For poliomyelitis: 1971 to 1980 (5803 cases and 1773 deaths), 1981 to 1990, (3685 cases and 521 deaths), 1991 to 2000 (2967 cases and 409 deaths) and from 2001 to 2010 (1371 cases and 21 deaths).

Conclusions: Outbreaks of epidemics have been recurrent and prolonged in Cameroon despite measures put in place by the Cameroon government and its partners. Improving hygiene, sensitization vaccination and surveillance and would be key in addressing these epidemics.

Toxicity Assessment of Biologically Degraded Product of Textile Dye Acid Maroon V

2026-06-01T13:33:02+00:00

Azo dyes are subjected to environmental concern as a result of their resistance to degradation. Some decolorized and degraded products of a variety of azo dyes are toxic and mutagenic to flora and fauna. The toxic activity of azo dyes is also dependent on the type and position of the aromatic rings and amino nitrogen atoms. Several reviews have been focusses so far on the biodegradation of azo dye pollutants, although the significance of their biodegraded product has not been evaluated. In view of this gap, we have studied the impact of degradation products of di-azo textile dye Acid Maroon V by novel bacterial isolate Bacillus subtilis C3. The HeLa cell line and human lymphocyte cells were employed to test the genotoxicity and cytotoxicity of Acid Maroon V and its breakdown products, respectively. The Acid Maroon V degradation products showed similar activity to its parent dye in MTT assay. However, when compared to the positive control mitomycin, neither the sister chromatid exchange assay nor the chromosome aberration assay revealed any appreciable chromosomal changes.

Antibacterial Activity of Green Synthesized Moringa oleifera-Mediated Silver Nanoparticles against Pseudomonas aeruginosa

2026-06-02T12:24:04+00:00

Background: Silver nanoparticles (AgNPs) have gained significant attention due to their remarkable antimicrobial properties and biomedical applications.

Aim: This study aimed to synthesize and characterize silver nanoparticles using Moringa oleifera leaf extract and evaluate their antibacterial activity against Pseudomonas aeruginosa.

Methods: Green synthesis of AgNPs was achieved using M. oleifera aqueous leaf extract as a reducing and stabilizing agent. The synthesized nanoparticles were characterized using UV-VIS Spectroscopy, X-ray diffraction (XRD), Fourier Transform Infrared (FT-IR) spectroscopy, and Scanning Electron Microscopy (SEM). Antibacterial activity was determined using agar well diffusion method.

Results: UV-VIS spectral study revealed that Moringa oleifera-Silver nanoparticles (MO-AgNPs) exhibited absorption band in the range of 267 nm. XRD analysis confirmed the crystalline nature of the nanoparticles with characteristic peaks at 2θ ≈ 30.31°, 38.16°, 44.30°, and 64.50° corresponding to face-centered cubic silver. FT-IR analysis revealed the presence of functional groups such as hydroxyl, amine, and carbonyl compounds involved in nanoparticle reduction and stabilization. SEM micrographs showed MO-AgNPs were spherical with an average of 50-60 nm Antibacterial activity revealed that the synthesized M. oleifera-AgNPs exhibited inhibitory activity against P. aeruginosa with an inhibition zone diameter of 13 mm, which was higher than that of ciprofloxacin (11 mm).

Conclusion: The findings indicates that Moringa oleifera-silver nanoparticles possess promising antibacterial potential and may serve as eco-friendly alternative antimicrobial agents for biomedical applications.