Emergence Of Multiresistant Bacteria Research Articles

Perception and practice of self-medication by parents on their children: A study carried out in the saint joseph health area of Kisangani (The democratic republic of Congo)

Introduction: Self-medication is a major public health problem in view of the risks involved, such as therapeutic failures and the emergence of multi-resistant bacteria. The aim of the study is to demonstrate how self-medication of children is perceived by parents living in the Saint Joseph Health Area, to identify the satisfaction that parents derive from self-medication, to determine the way in which these parents self-medicate their children, and to present what parents can expect from self-medication. Methodology: This was a qualitative, phenomenological study based on free, face-to-face interviews. It concerned 4 mothers found in pre-school consultations at the Saint Joseph Reference Health Centre from June 15 to 30, 2023. Results: With regard to perception, the respondents had demonstrated the significance of self-medication, the determining factors, and their experience on this subject by often resorting to old prescriptions and medicine leaflets. Low income and the time it takes to get to the hospital were the determining factors. The respondents found self-medication satisfying. They were aware of the dangers and risks associated with self-medication. Risk prevention consisted of giving the child palm oil and skimmed milk. Their expectation of self-medicating was always to be cured. Conclusion: Self-medication in Kisangani is a real phenomenon, with the majority of parents practicing it for themselves and their children due to a lack of financial means, as medical care is expensive. Free medical care would be one way of combating this practice.

Antibioprophylaxie chirurgicale et medicale courte de l’enfant

Antibiotic prophylaxis aims to counteract bacterial proliferation in order to reduce the risk of infection in a patient. The emergence of multi-resistant bacteria, which can lead to therapeutic impasses, has prompted the international medical community to regularly propose programs to optimize the use of antibiotics. In collaboration with the SFAR (Société Française d'Anesthésie Réanimation), the GPIP has updated its recommendations for good practice in surgical antibiotic prophylaxis in children, and for short antibiotic prophylaxis in medicine. These recommendations provide a general framework based on the analysis of national and international data, and more often than not on expert opinion, and are intended to support the implementation of local antibiotic prophylaxis protocols specific to each team.

Antibacterial Activities of Aqueous and Hydroethanolic Extracts of Anogeissus leiocarpus on the In vitro Growth of two Multiresistant Strains of Salmonella typhimurium Isolated from Broilers Chickens

Poultry products have quickly become one of the most consumed sources of protein in the world. However, poultry farmers face enormous economic losses caused by the emergence of multi-resistant bacteria. In addition, the consumption of eggs and poultry meat also causes several infectious diseases within populations as well as poisonings and therefore a public health problem.
 Aims: The objective of our study was to evaluate in vitro the antibacterial properties of aqueous and 70% hydroethanolic extracts of the leaves of Anogeissus leiocarpus on two strains of multiresistant Salmonella typhimurium isolated from the faeces of broilers chickens.
 Methodology: The agar well method was used to test the susceptibility of bacterial strains while the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined by the liquid dilution method.
 Results: On the two multiresistant Salmonella typhimurium germs, the aqueous and 70% hydroethanolic extracts of Anogeissus leiocarpus gave zones of inhibition diameter between 13-22 mm while the minimum bactericidal concentrations (MBC) were ranged between 12.5 mg/ml and 25 mg/ml. The results of the phytochemical analysis of the extracts studied reveal the presence of several families of secondary metabolites including phenolic compounds, cardiac glycosides, saponins, alkaloids and then sterols and terpenes. However, these secondary metabolites are distributed differently within the aqueous and 70% hydroethanolic extracts.
 Conclusion: In view of the results obtained in the present work, this plant could be used as a phytomedicine to combat pathologies linked to Salmonella typhimurium in broiler chickens and as an antibacterial agent in food products that may harbor strains of Salmonella typhimurium.

Methicillin-Resistant Staphylococcus aurous: Epidemiology, Transmission and New Alternative Therapies: A Narrative Review.

Over the last decade, we were facing medical struggle by the emergence of multi-resistant bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA). MRSA infections are still causing a growing global concern due to the rapid adaptive multidrug resistance to conventional antibiotics in human, community and veterinary medicine. Here we provide an overview about MRSA epidemiology, transmission and alternative potential treatments particularly new discovered phytochemicals with biological activity. In this narrative review, bibliographic data was collected from literature search databases: Google Scholar, web of science and PubMed/MEDLINE during recent years (2016 to 2021). MRSA is responsible of wide spectrum life threatening infections such us septicemia, endocarditis, and wound infections. It has epidemic potential in hospitals, that is responsible of most nosocomial infections leading to mortality and constitute a real burden for the healthcare systems. Effective preventive strategies for management of MRSA are highly required moreover, the identification and development of novel drugs or active biomolecules through phytochemicals are time challenging to face new resistant strains.

Prevalence of Esbl-Producing Enterobacteriaceae Strains Isolated to the University Hospital Center of N’djamena and their Sensitivity to Antibiotics

Introduction: The resistance of Enterobacteriaceae to antibiotics is experiencing a worrying worldwide evolution with a growing impact in the community and medical sector. The difficulties encountered in hospitals in treating certain infections resulting from multi-resistant bacteria are a source of great concern in human health. The aim of this study was to determine the prevalence, diversity and antibiotic resistance of Extended-Spectrum Beta-Lactamase (ESBL)-producing Enterobacteriaceae in the hospital setting. Materials and Methods: A total of 830 specimens from hospitalized and outpatients including 341 stools, 257 urine and 232 pus were collected between August 2017 and February 2019. Enterobacteriaceae were identified using the Api 20E gallery and the VITEK® 2TM 15 compact automaton. E. coli and Salmonella serotyping were performed by serum agglutination using the Latex Test Kil and the Kauffmann-White Kit, respectively. Sensitivity tests according to the Antibiogram Committee of the French Society of Microbiology and double disc synergy to detect ESBLs have been carried out. Results: Out of the 830 specimens, 181 (21%) strains were isolated at the N’Djamena National Reference University Hospital between August 2017 and February 2019. 56 strains produced ESBLs, i.e. a prevalence of 30.9% (56/181). Escherichia coli was more common (74%), followed by Klebsiella pneumoniae (6.6%), Proteus spp (14.9%), Salmonella (2.8%) and Shigella (1.7%) (p<0,05). These germs were isolated from urine at 47%, pus at 38% and stool at 15%. Resistance with E. coli was respectively 60.8% and 50% for imipenem and amoxicillin + clavulanic acid. Regarding Klebsiella pneumoniae, these rates were 70% and 55% respectively. Salmonella showed resistance to ciprofloxacin (80%), gentamicin (80%) and ceftriaxone (60%). E. coli, Klebsiella pneumoniae have been the most implicated with strong resistance to the majority of antibiotics. Conclusion: This study highlighted the diversity of enterobacteria causing diarrhea and the emergence of multi-resistant bacteria. It helps to update knowledge for better patient care.

The Chemical Space of Marine Antibacterials: Diphenyl Ethers, Benzophenones, Xanthones, and Anthraquinones.

The emergence of multiresistant bacteria and the shortage of antibacterials in the drug pipeline creates the need to search for novel agents. Evolution drives the optimization of the structure of marine natural products to act as antibacterial agents. Polyketides are a vast and structurally diverse family of compounds that have been isolated from different marine microorganisms. Within the different polyketides, benzophenones, diphenyl ethers, anthraquinones, and xanthones have shown promising antibacterial activity. In this work, a dataset of 246 marine polyketides has been identified. In order to characterize the chemical space occupied by these marine polyketides, molecular descriptors and fingerprints were calculated. Molecular descriptors were analyzed according to the scaffold, and principal component analysis was performed to identify the relationships among the different descriptors. Generally, the identified marine polyketides are unsaturated, water-insoluble compounds. Among the different polyketides, diphenyl ethers tend to be more lipophilic and non-polar than the remaining classes. Molecular fingerprints were used to group the polyketides according to their molecular similarity into clusters. A total of 76 clusters were obtained, with a loose threshold for the Butina clustering algorithm, highlighting the large structural diversity of the marine polyketides. The large structural diversity was also evidenced by the visualization trees map assembled using the tree map (TMAP) unsupervised machine-learning method. The available antibacterial activity data were examined in terms of bacterial strains, and the activity data were used to rank the compounds according to their antibacterial potential. This potential ranking was used to identify the most promising compounds (four compounds) which can inspire the development of new structural analogs with better potency and absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties.

Evaluation of self-medication habits among patients at the Odontostomatology unit of a secondary health facility in Cameroon

Self-medication is a major public health issue because of the hazard it creates, such as therapeutic failures and the emergence of multi-resistant bacteria, but it is still poorly documented in our local context.Our goal was to determine the prevalence of self-medication due oral diseases when they visit the Odontostomatology unit of a secondary health facility in Cameroon. Materials and methodsFrom February to May 2020, we conducted a descriptive cross-sectional study at the Odontostomatology unit of the PCC Dental Office of Djoungolo. After obtaining informed consent, data was collected using questionnaires. For the statistical analysis, the significance level was set at 5%. ResultsWe retained 147 patients (75 women and 72 men) in our study. The average age was 35.3 years ± 13.5 years. Self-medication was used by 88% of our patients, and the most common reason for consultation (81% of cases) was dental pain. The most used drugs for self-medication in our patients were NSAIDs and analgesics. In our study, amoxicillin was the most used antibiotic (21%). In terms of supply routes, the pharmacy was the most common source of drug supply (39%). In our study, the factors significantly associated with self-medication were age >30, low levels of education, and certain socio-professional activities. ConclusionThe prevalence of self-medication during oral diseases is high and is closely related to our country's socioeconomic context. This study highlights the primacy of educating patients about the risks of taking drugs without a prescription and the benefits of having routine consultations with an oral physician.

Doderlin: isolation and characterization of a broad-spectrum antimicrobial peptide from Lactobacillusacidophilus

Lactobacillus acidophilus are Gram-positive bacteria distributed in diverse environments, and as a component of the normal microbiota of gastrointestinal and urogenital tract, they are relevant for human beings. Classified as lactic acid bacteria, due to the production of lactic acid, Lactobacillus can also produce antimicrobial peptides (AMPs), which is a compound synthesized by all forms of life aiming for protecting themselves from threats and to increase their competitivity to survive in a specific environment. AMPs are molecules capable of inhibiting the growth of microorganisms and, due to the indiscriminate use of conventional antibiotics and the emergence of multi-resistant bacteria, they have become an alternative, not only for treating multi-resistant infections, but also for the identification of probiotic products and food conservation. Considering the rampant rise of bacterial resistance to classical antimicrobials, the present study aimed to isolate and characterize AMPs from L. acidophilus extracts. Lactobacillus acid extract was pre-fractionated on disposable cartridges, followed by a high-performance liquid chromatography (HPLC). The collected fractions were evaluated in a liquid growth inhibition assay allowing to identify eight fractions with antimicrobial activity, and one of them showed antimicrobial activity against Candida albicans and, for this reason, was further characterized by mass spectrometry (MS). A peptide with a molecular mass of 1788.01 Da, showing the primary sequence NEPTHLLKAFSKAGFQ, as determined by MS, was named as Doderlin. Interestingly, antimicrobial molecules isolated from L. acidophilus have already been described previously, but few reports describe AMPs effective against C. albicans as the one reported here. We show here that this newly discovered molecule has a biological property with potential to be used in pharmaceutical and food companies, in the fight against contamination and/or for treating infections caused by microorganisms, respectively. ImportanceDoderlin is a newly discovered molecule with a biological property with potential to be applied in pharmaceutical and food companies, in the fight against contamination and/or for treating infections caused by microorganisms, respectively.

Adsorption of Emerging Pollutant by Pecan Shell-Based Biosorbent

The insertion of antibiotics in water resources results from anthropogenic sources; however, at residual concentrations, they characterize potential risks to the ecosystem, such as the emergence of multi-resistant bacteria. It is necessary to develop technologies to provide sustainable solutions for low- and middle-income countries. Thus, the present study aims to evaluate the ability to remove the antibiotic ciprofloxacin (CPX) with a biosorbent produced with pecan shells (PSB). The PSB structure was determined by scanning electron microscopy and spectroscopy in the infrared region by Fourier Transform. For adsorption assays, solutions of 10 mg L−1 of CPX were used. The results show that the process reaches equilibrium at 240 min, and follows the pseudo-second order model kinetic and the Freundlich equilibrium model. The increase in temperature and the pH variation of the solution strongly influence the process. In general, the adsorption of CPX using PS is a potential method for treating water and contaminated effluents, as well as being a low-cost method; this is because it uses a byproduct from the agricultural industry that results in a reduction of approximately 60% of the antibiotic load contained in the liquid effluent.

Evaluation of the antibacterial activity of three types of Vernonia amygdalina Del. leaf extract sold in the markets of Lomé-Togo

Nowadays, the emergence of multi-resistant bacteria and the absence of new antibiotics are an increasing risk of therapeutic impasse. Nutraceuticals are plants which are reputed to have therapeutic properties and which are consumed as food. One of these plants consumed as a leafy vegetable, having amoebicidal properties and very popular among togolese populations is Vernonia amygdalina. The objective of this work was to evaluate the antibacterial activity of three types of V. amygdalina leaf extracts. The extracts were obtained by maceration of dried leaf powder (hydroethanolic 30:70), by decoction of fresh leaves and crushed boiled leaves (FEBEVA). The evaluation of the antibacterial activity of the extracts was carried out by the diffusion method and microdilution. The decoctate had a bactericidal effect on S. aureus and K. pneumoniae. FEBEVA was only activated on day 7 and only with a concentration of 400 mg. Phytochemical screening revealed secondary metabolites such as saponosides, tannins, triterpenes, polyphenols, sterols, alkaloids. This work has provided a scientific basis for the use of Vernonia amygdalina in traditional pharmacopoeia, particularly in the treatment of bacterial pathologies.

Bacteriophages as Antimicrobial Agents? Proteomic Insights on Three Novel Lytic Bacteriophages Infecting ESBL-Producing Escherichia coli.

With the emergence of multiresistant bacteria, the use of bacteriophages is gaining renewed interest as potential antimicrobial agents. The aim of this study was to analyze the structure of three lytic bacteriophages infecting Escherichia coli (SD1, SD2, and SD3) using a gel-based proteomics approach and the cellular response of this bacterium to phage SD1 infection at the proteome level. The combination of the results of 1-DE and 2-DE followed by mass spectrometry led to the identification of 3, 14, and 9 structure proteins for SD1, SD2, and SD3 phages, respectively. Different protein profiles with common proteins were noticed. We also analyzed phage-induced effects by comparing samples from infected cells to those of noninfected cells. We verified important changes in E. coli proteins expression during phage SD1 infection, where there was an overexpression of proteins involved in stress response. Our results indicated that viral infection caused bacterial oxidative stress and bacterial cells response to stress was orchestrated by antioxidant defense mechanisms. This article makes an empirical scientific contribution toward the concept of bacteriophages as potential antimicrobial agents. With converging ecological threats in the 21st century, novel approaches to address the innovation gaps in antimicrobial development are more essential than ever. Further research on bacteriophages is called for in this broader context of planetary health and integrative biology.

Frontispiece: Handling (Nano)Silver as Antimicrobial Agent: Therapeutic Window, Dissolution Dynamics, Detection Methods and Molecular Interactions

Silver is a well-known antimicrobial and with the emergence of multiresistant bacteria it has come back into the focus of research; ionic as well as nano-sized silver have been studied in vitro and in vivo. The results are controversial, silver being discussed as the “silver bullet” or a “wolf in sheep's clothing”. This Review aims to give insight into the activity and mechanism of silver in biological media, be it in contact with bacteria or cells. For more details see the Review by K. M. Fromm et al. on page 10948 ff.

Handling (Nano)Silver as Antimicrobial Agent: Therapeutic Window, Dissolution Dynamics, Detection Methods and Molecular Interactions.

Silver is an antimicrobial agent well known since antiquity. With the emergence of multiresistant bacteria, it has come back into the focus of research, and ionic as well as nano-sized silver have been studied in vitro and in vivo. The results are controversial, silver being discussed as the "silver bullet" or a "wolf in sheep's clothing". A thorough search of literature from chemistry, materials and environmental science, biology and medicine led to this Review which summarizes the potential use of silver and its compounds in medicine, ongoing processes of dissolution and the different methods by which this usefulness can be evaluated. It also highlights the therapeutic window of silver, mechanistic interactions of silver and biological media as well as best practices for handling silver in a biomedical environment. This Review reflects the current knowhow and observations, and may thus give hints and guidelines to understand and interpret the observed effects.

Activity of five antimicrobial peptides against periodontal as well as non-periodontal pathogenic strains

ABSTRACT Background: Due to the increasing emergence of multi-resistant bacteria the search for alternative antimicrobial substances is of high interest. Promising agents are antimicrobial peptides which are host defense molecules of the innate immune system in a wide range of different species. Objectives: The aim of this study was to assess the activity of nisin, melittin, lactoferrin, parasin-1 and LL-37 against 35 oral bacteria and Candida albicans employing the gold standard method for anaerobic susceptibility testing. Methods: The activity of the peptides was determined by an agar dilution method under anaerobic and aerobic conditions. The test media contained final peptide concentrations between 0.125 µg/ml and 8 µg/ml (melittin, lactoferrin, parasin-1, LL-37) and between 0.125 µg/ml and 128 µg/ml (nisin). Results: Nisin completely inhibited the growth of Megasphaera sp., Bifidobacterium longum, Parvimonas micra, Actinomyces israelii, Actinomyces naeslundii, Actinomyces odontolyticus, Prevotella intermedia, Streptococcus anginosus, Streptococcus constellatus and Staphylococcus aureus. Melittin and lactoferrin reduced the growth of Megasphaera sp., P. micra, B. longum (melittin) and Selenomonas flueggei (lactoferrin). Parasin-1 and LL-37 showed no activity. Conclusion: AMPs, especially nisin and to a smaller degree lactoferrin, might be promising alternatives to antibiotics because of their antimicrobial activity, high resistance to environmental conditions and partially low costs.

Rapid antimicrobial susceptibility testing on positive blood cultures through an innovative light scattering technology: performances and turnaround time evaluation

BackgroundA bacteremia diagnosis with speeded-up identification and antimicrobial susceptibility testing (AST) is mandatory to adjust empirical broad-spectrum antibiotherapy and avoid the emergence of multi-resistant bacteria. Alfred 60AST (Alifax, Polverara, PD, Italy) is an innovative automated system based on light scattering measurements allowing direct AST from positive blood cultures with rapid results. In this study we aimed to evaluate the system’s performances and turnaround time (TAT) compared to routine AST.MethodsThe study was conducted during 2 non-consecutive 3-month periods at the microbiology laboratory of the Cliniques universitaires Saint-Luc. All blood cultures detected positive in the 0 AM–10 AM time frame with a pure Gram-positive cocci or Gram-negative bacilli stain were included for Alfred 60AST testing. Two customized EUCAST antibiotic panels were set up composed of 1) a “Gram-negative” panel including cefuroxime, ceftazidime Enterobacteriaceae, piperacillin-tazobactam Enterobacteriaceae, ciprofloxacine, and ceftazidime Pseudomonas 2) a “Gram-positive” panel including cefoxitin Staphylococcus aureus, cefoxitin coagulase-negative (CNS) Staphylococci and ampicillin Enterococci. Categorical agreement (CA), very major errors (VME), major errors (ME), minor errors (mE) and TAT to Alfred 60AST results were calculated in comparison with AST results obtained from direct testing on positive blood cultures with the Phoenix system (Becton Dickinson, Franklin Lakes, NJ, USA).ResultsFive hundred seventy and one hundred nine antibiotics were evaluated on respectively 166 Gram-negative bacilli and 109 Gram-positive cocci included in the studied population. During the first study period regarding Gram-negative strains a CA of 89.5% was obtained with a high rate of VME (19 and 15.4% respectively) for cefuroxime and piperacillin-tazobactam Enterobacteriaceae. Considering this, Alifax reviewed these antibiotics’ formulations improving Gram-negative bacilli total CA to 92.2% with no VME during the second study period. For Gram-positive cocci, total CA was 88.1% with 2.3% VME, 13.8% ME (mainly cefoxitin CNS) and 12% mE rates both study periods combined. Median TAT to AST results was 5 h with Alfred versus 12 h34 with Phoenix.ConclusionThe Alfred 60AST system shows correct yet improvable microbiological performances and a major TAT reduction compared to direct automated AST testing. Clinical studies measuring the impact of the approach on antibiotic management of patients with bacteremia are recommended.

Bacteria Sensors Based on Interdigitated Silicon Nanowires Electrodes

The detection of pathogenic bacteria in food, water and air is an important issue because of its critical impact on public health, especially with the emergence of multi-resistant bacteria. Standard microbiological methods of bacterial culture impose deadlines that can slow down the establishment of means of protocols against contaminations. In this context, a strategy is to develop ultrasensitive sensors based on silicon nanowires networks for rapid detection of bacteria. Indeed, the nanowires have a large contact surface allowing preferential interactions with the bacteria. Resistors based on silicon nanowires used as bacteria sensors are fabricated using conventional silicon technologies. The nanowires are obtained by the VLS (Liquid Solid Vapor) method using gold as catalyst and the interdigited comb electrodes are made of high doped polycrystalline silicon. The bacteria are preferentially attached to the nanowire network (fig. 1a and b). The electrical characterization of the the devices shows that the current through the resistors increases significantly due to the presence of bacteria (fig. 1c). The results show the potential use of the resistors, with the nanowires as sensitive units, for the detection of bacteria. Figure 1

Antibio-gouvernance en néonatalogie

Rational use of antibiotic has become a national and international health priority to fight against the emergence of multiresistant bacteria. Neonates are particularly exposed to antibiotic treatments because of their high susceptibility to severe infection and the lack of specificity of sepsis signs that make diagnosis difficult. This population is also particularly susceptible to microbiota disruption due to antibiotic treatment. Implementation of antibiotic stewardship in neonate is then an urgent need. According to a literature review, principles of antibiotic stewardship implementation in neonate are based on: (i) a multidisciplinary team comprising infectious disease specialists and aware of neonatal specificities (diagnosis, bacterial epidemiology, pharmacology) ; (ii) clear and easy-to-evaluate goals discussed a priori with neonatologists ; (iii) short-term assessment of the impact on antibiotic consumption and antimicrobial resistance ; (iv) enablement of the healthcare professionals within the ward to enhance the sustainability and (v) support from the institution.

Alcohol consumption and risk of infection after a variceal bleeding in low-risk patients.

Antibiotic prophylaxis is a cornerstone in the management of acute variceal bleeding. However, emergence of multiresistant bacteria and antibiotic-associated complications is a growing problem in cirrhosis. It has been proposed that certain low-risk populations may have good outcomes without antibiotic. We aimed to analyse the stratified risk of bacterial infection after a variceal bleeding in previously considered low-risk patients. We analysed prospective data from all consecutive cirrhotic patients with acute variceal bleeding admitted to our tertiary hospital between 2004 and 2012. All patients received somatostatin, variceal ligation and antibiotic prophylaxis. Patients were followed until day 42 or death. Patients were stratified based on Child-Pugh class. Within the low-risk strata, further one-step stratification was performed using baseline risk factors identified through logistic regression multivariate adjustment. Two hundred and fifteen patients were included. Twenty-seven patients (12.5%) developed 32 bacterial infections within 6 weeks after the index bleeding. Multivariate adjustment identified alcohol consumption as a significant risk factor for infection. Within previously considered low-risk patients (Child-Pugh A), the risk of infection was significantly higher in patients with active alcohol consumption (21.4% vs. 0% in non-drinkers, P = 0.015). The risk of infection in Child-Pugh A and B patients with non-alcohol cirrhosis receiving antibiotics was very low (2/81, 2.5%). Alcohol consumption significantly increased the risk of infection after a variceal bleeding in previously considered low-risk patients. Within Child-Pugh A class, patients with active alcohol consumption should not be considered at low risk of infection.

Risk factors for colonization and infection by multiresistant bacteria

In 2013, the “Zero Resistance” (RZ) program was launched in Spain, to help prevent the emergence of multiresistant bacteria (MRB) in critically ill patients. One of its recommendations is to complete a checklist upon patient admission in Intensive Care Unit (ICU) to identify those patients at high risk for colonization or infection by MRB*.

Characteristics of isolated multiresistant bacteria according germ, location and “zero resistance” criteria

Multiresistant bacteria (MRB) development is a growing phenomenon. In 2013, the “Zero Resistance” (RZ) program was launched in Spain, to help prevent the emergence of multiresistant bacteria (MRB) in critically ill patients. One of its recommendations is to complete a checklist upon patient admission in Intensive Care Unit (ICU) to identify those patients at high risk for colonization or infection by MRB*.