Effect of free ammonia concentration on monochloramine penetration within a nitrifying biofilm and its effect on activity, viability, and recovery

Chloramine has replaced free chorine for secondary disinfection at many water utilities because of disinfection by-product (DBP) regulations. Because chloramination provides a source of ammonia, there is a potential for nitrification when using chloramines. Nitrification in drinking water distribution systems is undesirable and may result in degradation of water quality and subsequent non-compliance with existing regulations. Thus, nitrification control is a major issue and likely to become increasingly important as chloramine use increases. In this study, monochloramine penetration and its effect on nitrifying biofilm activity, viability, and recovery was investigated and evaluated using microelectrodes and confocal laser scanning microscopy (CLSM). Monochloramine was applied to nitrifying biofilm for 24 h at two different chlorine to nitrogen (Cl₂:N) mass ratios (4:1 [4.4 mg Cl₂/L] or 1:1 Cl₂:N [5.3 mg Cl₂/L]), resulting in either a low (0.23 mg N/L) or high (4.2 mg N/L) free ammonia concentration. Subsequently, these biofilm samples were allowed to recover without monochloramine and receiving 4.2 mg N/L free ammonia. Under both monochloramine application conditions, monochloramine fully penetrated into the nitrifying biofilm within 24 h. Despite this complete monochloramine penetration, complete viability loss did not occur, and both biofilm samples subsequently recovered aerobic activity when fed only free ammonia. When monochloramine was applied with a low free ammonia concentration, dissolved oxygen (DO) fully penetrated, but with a high free ammonia concentration, complete cessation of aerobic activity (i.e., oxygen utilization) did not occur and subsequent analysis indicated that oxygen consumption still remained near the substratum. During the ammonia only recovery phase, different spatial recoveries were seen in each of the samples, based on oxygen utilization. It appears that the presence of higher free ammonia concentration allowed a larger biomass to remain active during monochloramine application, particularly the organisms deeper within the biofilm, leading to faster recovery in oxygen utilization when monochloramine was removed. These results suggest that limiting the free ammonia concentration during monochloramine application will slow the onset of nitrification episodes by maintaining the biofilm biomass at a state of lower activity.     

Measurement of antibiotic resistance of microbial species in the Karasu River (Turkey) using molecular techniques

The Karasu River (Sinop, Turkey) was sampled for bacteriological analyses and investigation of antibiotic resistance at the bacterial isolates. In the study, the physical properties and numbers of total aerobic (TA), total coliform (TC), faecal coliform (FC), faecal Streptococci (FS) and Escherichia coli in water samples were investigated. E. coli strains were subjected to antibiotic susceptibility testing, antibiotic resistant genes, virulence genes and plasmid DNA analysis and fingerprinting techniques. The bacteriological analysis results showed that the difference between TA, TC, FC and FS numbers (p > 0.05) was not significant for a total of four stations. The Multiple Antibiotic Resistance Index (MARI) values of tested strains were found in range from 0.4 to 0.7. The antibiotic resistance genes of strains; tetA (33.9%), bla_cym‐2 (12.5%) and cat1 (16.7%) were screened by PCR method. Out of a total of 57 E. coli isolates, 11 E. coli strains were positive for the eae (19.6%) gene, representing atypical EPEC, whilst only one strain was positive for the eae and stx1 (1.7%) gene, representing STEC. Present study has shown that the aquatic environments can be reservoirs of pathogenic E. coli strains. The study also indicated that PCR‐based techniques might especially be helpful in the rapid identification of multidrug‐resistance analysis and virulence genes of strains. Karasu stream, which is important in Sinop province, Turkey, was investigated for the first time in this study in terms of a molecular microbiological perspective.     

Oxidation of sulfhydryl groups by monochloramine

In recent years there has been an increased used of monochloramine (NH₂Cl) for water disinfection because of its low trihalomethane formation potential. Monochloramine is also the predominant disinfectant upon chlorination of wastewater effluents. In an effort to more clearly understand the disinfectant's mode of action in inactivating microorganisms, a study was undertaken to evaluate the compound's reactions with sulfhydryl (−SH) groups. The extent of oxidation of these groups was dependent upon the molar ratio of −SH to NH₂Cl. When this ratio was >2:1, the reaction was reversible and ceased at disulfide formation. However, at a ratio of < 2:1, the reaction proceeded irreversibly beyond the disulfide; this reaction continued in the presence of a monochloramine residual. Not all −SH groups in Escherichia coli B were available for reaction. Masking of these groups within bacterial proteins prevented their complete oxidation at monochloramine doses as high as 100 mg 1⁻¹. The extent to which sulfhydryls are oxidized in bacteria may play an important role in further research on microbial reactivation.     

Modeling monochloramine loss in the presence of natural organic matter

A comprehensive model describing monochloramine loss in the presence of natural organic matter (NOM) is presented. The model incorporates simultaneous monochloramine autodecomposition and reaction pathways resulting in NOM oxidation. These competing pathways were resolved numerically using an iterative process evaluating hypothesized reactions describing NOM oxidation by monochloramine under various experimental conditions. The reaction of monochloramine with NOM was described as biphasic using four NOM specific reaction parameters. NOM pathway 1 involves a direct reaction of monochloramine with NOM (k_doc1=1.05×10⁴-3.45×10⁴ M⁻¹ h⁻¹). NOM pathway 2 is slower in terms of monochloramine loss and attributable to free chorine (HOCl) derived from monochloramine hydrolysis (k_doc2=5.72×10⁵-6.98×10⁵ M⁻¹ h⁻¹), which accounted for the majority of monochloramine loss. Also, the free chlorine reactive site fraction in the NOM structure was found to correlate to specific ultraviolet absorbance at 280 nm (SUVA₂₈₀). Modeling monochloramine loss allowed for insight into disinfectant reaction pathways involving NOM oxidation. This knowledge is of value in assessing monochloramine stability in distribution systems and reaction pathways leading to disinfection by-product (DBP) formation.     

Novel homologs of the multiple resistance regulator marA in antibiotic-contaminated environments

Antibiotics are commonly detected in the environment as contaminants. Exposure to antibiotics may induce antimicrobial-resistance, as well as the horizontal transfer of resistance genes in bacterial populations. We selected the resistance gene marA, mediating resistance to multiple antibiotics, and explored its distribution in sediment and water samples from surface and sewage treatment waters. Ciprofloxacin and ofloxacin (fluoroquinolones), sulphamethoxazole (sulphonamide), erythromycin, clarythromycin, and spiramycin (macrolides), lincomycin (lincosamide), and oxytetracycline (tetracycline) were measured in the same samples to determine antibiotic contamination. Bacterial populations from environmental samples were challenged with antibiotics to identify resistant isolates. The gene marA was found in almost all environmental samples and was confirmed by PCR amplification in antibiotic-resistant colonies. 16S rDNA sequencing revealed that the majority of resistant isolates belonged to the Gram-positive genus Bacillus, not previously known to possess the regulator marA. We assayed the incidence of marA in environmental bacterial populations of Escherichia coli and Bacillus by quantitative real-time PCR in correlation with the levels of antibiotics. Phylogenetic analysis indicated the possible lateral acquisition of marA by Bacillus from Gram-negative Enterobacteriaceae revealing a novel marA homolog in Bacillus. Quantitative PCR assays indicate that the frequency of this gene in antropised environments seems to be related to bacterial exposure to water-borne antibiotics.     

Efficiency of water disinfectants against Legionella pneumophila and Acanthamoeba

Free-living amoebae might be pathogenic by themselves and be a reservoir for bacterial pathogens, such as Legionella pneumophila. Not only could amoebae protect intra-cellular Legionella but Legionella grown within amoebae could undergo physiological modifications and become more resistant and more virulent. Therefore, it is important to study the efficiency of treatments on amoebae and Legionella grown within these amoebae to improve their application and to limit their impact on the environment.With this aim, we compared various water disinfectants against trophozoites of three Acanthamoeba strains and L. pneumophila alone or in co-culture. Three oxidizing disinfectants (chlorine, monochloramine, and chorine dioxide) were assessed. All the samples were treated with disinfectants for 1 h and the disinfectant concentration was followed to calculate disinfectant exposure (Ct). We noticed that there were significant differences of susceptibility among the Acanthamoeba strains. However no difference was observed between infected and non-infected amoebae. Also, the comparison between the three disinfectants indicates that monochloramine was efficient at the same level towards free or co-cultured L. pneumophila while chlorine and chlorine dioxide were less efficient on co-cultured L. pneumophila. It suggests that these disinfectants should have different modes of action. Finally, our results provide for the first time disinfectant exposure values for Acanthamoeba treatments that might be used as references for disinfection of water systems.     

Influence of tetracycline resistance on the transport of manure-derived Escherichia coli in saturated porous media

In this research, tetracycline resistant (tet^R) and tetracycline susceptible (tet^S) Escherichia coli isolates were retrieved from dairy manure and the influence of tetracycline resistance on the transport of E. coli in saturated porous media was investigated through laboratory column transport experiments. Experimental results showed that tet^RE. coli strains had higher mobility than the tet^S strains in saturated porous media. Measurements of cell surface properties suggested that tet^RE. coli strains exhibited lower zeta potentials than the tet^S strains. Because the surface of clean quartz sands is negatively charged, the repulsive electrostatic double layer (EDL) interaction between the tet^R cells and the surface of sands was stronger and thus facilitated the transport of the tet^R cells. Although no difference was observed in surface acidity, cell size, lipopolysaccharides (LPS) sugar content and cell-bound protein levels between the tet^R and tet^S strains, they displayed distinct outer membrane protein (OMP) profiles. It was likely that the difference in OMPs, some potentially related to drug efflux pumps, between the tet^R and tet^S strains led to alteration in cell surface properties which in turn affected cell transport in saturated porous media. Findings from this research suggested that manure-derived tet^RE. coli could spread more widely in the groundwater system and pose serious public health risks.     

Populations of antibiotic-resistant coliform bacteria change rapidly in a wastewater effluent dominated stream

Incomplete elimination of bacteria and pharmaceutical drugs during wastewater treatment results in the entry of antibiotics and antibiotic-resistant bacteria into receiving streams with effluent inputs. In Mud Creek in Fayetteville, AR, ofloxacin, trimethoprim, and sulfamethoxazole have been detected in water and sediment, and tetracycline has been detected in sediment downstream of treated effluent input. These antibiotics have been measured repeatedly, but at low concentrations (< 1 μg/L) in the stream. To determine if effluent input results in detectable and stable changes in antibiotic resistances downstream of effluent input, antibiotic resistance in Escherichia coli and total coliform bacteria in Mud Creek stream water and sediment were determined using a culture-based method. Isolated E. coli colonies were characterized for multiple antibiotic resistance (MAR) patterns on solid media and to evaluate E. coli isolate richness by amplification of a partial uidA gene followed by denaturant gradient gel electrophoresis (DGGE). Despite temporal variability, proportions of antibiotic-resistant E. coli were generally high in effluent and 640 m downstream. The MAR pattern ampicillin-trimethoprim-sulfamethoxazole was associated with a DGGE profile that was detected in effluent and downstream E. coli isolates, but not upstream. Percent resistance among coliform bacteria to trimethoprim and sulfamethoxazole was higher 640 m downstream compared to upstream sediment and water (with one exception). Resistance to ofloxacin was too low to analyze statistically and tetracycline resistance was fairly constant across sites. Resistances changed from 640 m to 2000 m downstream, although dissolved nutrient concentrations within that stream stretch resembled effluent. Antibiotic resistant bacteria are entering the stream, but resistances change within a short distance of effluent inputs, more quickly than indicated based on chemical water properties. Results illustrate the difficulty in tracking the input and fate of antibiotic resistance and in relating the presence of low antibiotic concentrations to selection or persistence of antibiotic resistances.     

High diversity and abundance of antibiotic-resistant Escherichia coli isolated from humans and farm animal hosts in Jeonnam Province, South Korea

The spread of antibiotics resistance among bacteria is a threat to human health. Since South Korea uses approximately 1.5 times more antibiotics than do other OECD countries, this is likely to impact the numbers and types of antibiotic-resistant bacteria found in the environment. In this study we examined feces from domesticated animals and humans for the diversity and abundance of antibiotic-resistant Escherichia coli. Abundant antibiotic-resistant E. coli were isolated from all the tested animals and humans and were examined by horizontal, fluorophore-enhanced, rep-PCR (HFERP) DNA fingerprint analysis. A total of 793 unique, non-clonal, E. coli isolates were obtained from the 513 human and animal hosts examined. Antibiotic resistance analysis, done using 14 antibiotics, indicated that 72.3% of the isolates (573 of 793) were found resistant to more than one antibiotic. The E. coli isolated from swine were resistant to the greatest number of antibiotics. Tetracycline resistant E. coli were routinely isolated from all animal hosts (36 to 77% per host), except for dairy cattle (9.3%). Twenty nine E. coli isolates from all hosts, except for duck, were resistant to more than 10 antibiotics. Gene transfer and southern hybridization studies revealed that resistance to 13 of the antibiotics was self-transmissible, and likely mediated by plasmids and integrons. Since genetically diverse and numerically abundant antibiotic-resistant E. coli were consistently recovered from chicken, swine and other domesticated animals in South Korea, our results suggest that the use of sub-therapeutic levels of antibiotics for disease prophylaxis and growth promotion should be curtailed.     

Antibiotic resistant enterococci and staphylococci isolated from flies collected near confined poultry feeding operations

Use of antibiotics as feed additives in poultry production has been linked to the presence of antibiotic resistant bacteria in farm workers, consumer poultry products and the environs of confined poultry operations. There are concerns that these resistant bacteria may be transferred to communities near these operations; however, environmental pathways of exposure are not well documented. We assessed the prevalence of antibiotic resistant enterococci and staphylococci in stored poultry litter and flies collected near broiler chicken houses. Drug resistant enterococci and staphylococci were isolated from flies caught near confined poultry feeding operations in the summer of 2006. Susceptibility testing was conducted on isolates using antibiotics selected on the basis of their importance to human medicine and use in poultry production. Resistant isolates were then screened for genetic determinants of antibiotic resistance. A total of 142 enterococcal isolates and 144 staphylococcal isolates from both fly and poultry litter samples were identified. Resistance genes erm(B), erm(A), msr(C), msr(A/B) and mobile genetic elements associated with the conjugative transposon Tn916, were found in isolates recovered from both poultry litter and flies. Erm(B) was the most common resistance gene in enterococci, while erm(A) was the most common in staphylococci. We report that flies collected near broiler poultry operations may be involved in the spread of drug resistant bacteria from these operations and may increase the potential for human exposure to drug resistant bacteria.     

Interactions of nanosilver with Escherichia coli cells in planktonic and biofilm cultures

Biofilms are often more resistant to toxic chemicals such as heavy metals and antimicrobial agents than planktonic cells. Nanosilver has a broad range of applications with strong antimicrobial activity. However, biofilm susceptibility to nanosilver toxicity is not well understood. We studied the bacterial activity in planktonic or biofilm cultures after nanosilver exposure using oxygen quenching fluorescence-based microrespirometry. We also determined the aggregation behavior and the spatial distribution of nanosilver having red fluorescence in biofilms of Escherichia coli expressing green fluorescent protein. At the same bacterial concentrations (3 × 10⁸ CFU/mL), biofilms were about four times more resistant to nanosilver inhibition than planktonic cells. The minimum bactericidal concentrations (MBCs) of nanosilver (size from 15 to 21 nm), defined as the lowest concentration that kills at least 99.9% of a planktonic or biofilm bacterial population, were 38 and 10 mg/L Ag, respectively. For comparison, silver ions were more toxic to E. coli than nanosilver with MBCs of 2.4 and 1.2 mg/L Ag for planktonic and biofilm cultures, respectively. Nanosilver was aggregated in the presence of planktonic or biofilm-forming cells resulting in an increase of average particle size by a factor of 15 and 40, respectively. The nanosilver particles were able to penetrate to approximately 40 μm in a thick biofilm after 1-h exposure. These findings suggested that biofilm resistance to nanosilver could be at least partially due to nanoparticle aggregation and retarded silver ion/particle diffusion.     

Antibiotic resistance in coagulase negative staphylococci isolated from wastewater and drinking water

This study reports the antibiotic resistance patterns of coagulase negative staphylococci (CNS) isolated from a drinking water treatment plant (WTP), a drinking water distribution network, responsible for supplying water to the consumers (WDN), and a wastewater treatment plant (WWTP), responsible for receiving and treating domestic residual effluents. Genotyping and the 16S rRNA gene sequence analysis demonstrated a higher diversity of species both in the WTP (6 species/19 isolates) and WWTP (12 species/47 isolates) than in the WDN (6 species/172 isolates). Staphylococcus pasteuri and Staphylococcus epidermidis prevailed in the WTP and WDN and Staphylococcus saprophyticus in the WWTP. Staphylococci with reduced susceptibility (resistance or intermediary phenotype) to beta-lactams, tetracycline, clindamycin and erythromycin were observed in all types of water and belonged to the three major species groups. The highest resistance rate was found against erythromycin, presumably due to the presence of the efflux pump encoded by the determinant msrA, detected in the majority of the resistant isolates. This study demonstrates that antibiotic resistant CNS may colonize different types of water, namely drinking water fulfilling all the quality standards.     

Association and decontamination of Bacillus spores in a simulated drinking water system

The objective of this work was to elucidate the disinfectant susceptibility of Bacillus anthracis Sterne (BA) and a commercial preparation of Bacillus thuringiensis (BT) spores associated with a simulated drinking water system. Biofilms composed of indigenous water system bacteria were accumulated on copper and polyvinyl chloride (PVC) pipe material surfaces in a low-flow pipe loop and uniformly mixed tank reactor (CDC biofilm reactor). Application of a distributed shear during spore contact resulted in approximately a 1.0 and 1.6 log₁₀ increase in the number of spores associated with copper and PVC surfaces, respectively. Decontamination of spores in both free suspension and after association with biofilm-conditioned pipe materials was attempted using free chlorine and monochloramine. Associated spores required 5- to 10-fold higher disinfectant concentrations to observe the same reduction of viable spores as in suspension. High disinfectant concentrations (103 mg/L free chlorine and 49 mg/L monochloramine) yielded less than a 2-log₁₀ reduction in viable associated spores after 60 min. Spores associated with biofilms on copper surfaces consistently yielded higher Ct values than PVC.     

Injury and repair of Escherichia coli damaged by acid mine water

Pure culture suspensions of Escherichia coli B/5 were stressed by exposure to filter-sterilized acid mine water (AMW). Sublethally injured survivors were examined for their ability to repair in several resuscitation media under different conditions of pH, temperature and oxygen availability. The repair process was monitored as a function of time by periodically removing samples from the repair media and simultaneously plating on nonselective and selective media. E. coli was severely damaged by AMW; however, sublethally injured survivors repaired when placed under favorable conditions. Optimal repair occurred in trypticase soy broth supplemented with 0.3% yeast extract (TSYB) at pH 7.0 and 35°C. Resuscitation did not occur in TSYB at pH 9.0, at an incubation temperature of 20°C, or in the absence of oxygen. Lauryl tryptose broth (LTB), which is recommended for the presumptive isolation of fecal coliforms, was unable to facilitate repair of injury. The presence of the surfactant, sodium dodecyl sulfate, as well as the nutrient composition of LTB, appeared to be responsible for the inability of this medium to permit recovery of AMW-stressed E. coli.     

Cohesiveness and hydrodynamic properties of young drinking water biofilms

Drinking water biofilms are complex microbial systems mainly composed of clusters of different size and age. Atomic force microscopy (AFM) measurements were performed on 4, 8 and 12 weeks old biofilms in order to quantify the mechanical detachment shear stress of the clusters, to estimate the biofilm entanglement rate ξ. This AFM approach showed that the removal of the clusters occurred generally for mechanical shear stress of about 100 kPa only for clusters volumes greater than 200 μm³. This value appears 1000 times higher than hydrodynamic shear stress technically available meaning that the cleaning of pipe surfaces by water flushing remains always incomplete. To predict hydrodynamic detachment of biofilm clusters, a theoretical model has been developed regarding the averaging of elastic and viscous stresses in the cluster and by including the entanglement rate ξ. The results highlighted a slight increase of the detachment shear stress with age and also the dependence between the posting of clusters and their volume. Indeed, the experimental values of ξ allow predicting biofilm hydrodynamic detachment with same order of magnitude than was what reported in the literature. The apparent discrepancy between the mechanical and the hydrodynamic detachment is mainly due to the fact that AFM mechanical experiments are related to the clusters local properties whereas hydrodynamic measurements reflected the global properties of the whole biofilm.     

Species association increases biofilm resistance to chemical and mechanical treatments

The study of biofilm ecology and interactions might help to improve our understanding of their resistance mechanisms to control strategies. Concerns that the diversity of the biofilm communities can affect disinfection efficacy have led us to examine the effect of two antimicrobial agents on two important spoilage bacteria. Studies were conducted on single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens. Biofilms were formed on a stainless steel rotating device, in a bioreactor, at a constant Reynolds number of agitation (Re_A). Biofilm phenotypic characterization showed significant differences, mainly in the metabolic activity and both extracellular proteins and polysaccharides content. Cetyl trimethyl ammonium bromide (CTAB) and glutaraldehyde (GLUT) solutions in conjunction with increasing Re_A were used to treat biofilms in order to assess their ability to kill and remove biofilms. B. cereus and P. fluorescens biofilms were stratified in a layered structure with each layer having differential tolerance to chemical and mechanical stresses. Dual species biofilms and P. fluorescens single biofilms had both the highest resistance to removal when pre-treated with CTAB and GLUT, respectively. B. cereus biofilms were the most affected by hydrodynamic disturbance and the most susceptible to antimicrobials. Dual biofilms were more resistant to antimicrobials than each single species biofilm, with a significant proportion of the population remaining in a viable state after exposure to CTAB or GLUT. Moreover, the species association increased the proportion of viable cells of both bacteria, comparatively to the single species scenarios, enhancing each other's survival to antimicrobials and the biofilm shear stress stability.     

Control of biofilm formation in water using molecularly capped silver nanoparticles

Control of biofouling and its negative effects on process performance of water systems is a serious operational challenge in all of the water sectors. Molecularly capped silver nanoparticles (Ag-MCNPs) were used as a pretreatment strategy for controlling biofilm development in aqueous suspensions using the model organism Pseudomonas aeruginosa. Biofilm control was tested in a two-step procedure: planktonic P. aeruginosa was exposed to the Ag-MCNPs and then the adherent biofilm formed by the surviving cells was monitored by applying a model biofilm-formation assay. Under specific conditions, Ag-MCNPs retarded biofilm formation, even when high percentage of planktonic P. aeruginosa cells survived the treatment. For example, Ag-MCNPs (10 μg mL⁻¹) retarded biofilm formation (>60%), when 50 percent of the planktonic P. aeruginosa cells survived the treatment. Moreover, stable low value of relative biomass has been formed in the presence of fixed Ag-MCNPs concentrations at various biofilm incubation times. Our results showed that Ag-MCNPs pretreated cells were able to produce EPS although they succeeded to form relatively low adherent biofilm. These pretreated cells appear well preserved and undamaged under TEM HPH/freeze micrographs, yet the intra cellular material seems to be pushed towards the peripheral parts of the cell, possibly indicating a survival strategy to the presence of Ag-MCNPs. The lower value of relative biomass formed in the presence of Ag-MCNPs could be associated with molecular mechanisms related to biofilm formation or continuous release of silver ions in the sample. However, further research is required to examine these factors.     

Pulsed ultraviolet light inactivation of Pseudomonas aeruginosa and Staphylococcus aureus biofilms

Microbial biofilms are complex communities that form when planktonic bacterial species attach to surfaces in many settings where they can provide a source of pathogenicity. The relative ineffectiveness of conventional disinfectants such as free chlorine and monochloramine for the inactivation of some species found in water has led to evaluation of alternative disinfectants for drinking and wastewater treatment. In recent years, novel pulsed power electrotechnologies have been introduced and are being considered as possible alternatives to current methods for inactivating problematic species in water. This study focuses on the ultraviolet (UV) inactivation of bacterial biofilms using a pulsed UV light approach as a potential disinfection method for water treatment operating systems. Biofilms were stimulated to form attached to polyvinyl chloride coupons using a recommended Centre for Disease Control biofilm reactor followed by exposure to a range of UV doses. Findings show that this method is highly effective at inactivating both planktonic and biofilm cells with significant inactivation rates obtained for both test species. Specifically, a 7.2 and 5.9 log₁₀ inactivation was achieved with up to 21.6 μJ/cm² UV for Pseudomonas aeruginosa and Staphylococcus aureus, respectively. Findings from this study highlight the effectiveness of pulsed UV for the inactivation of Pseudomonas biofilms among other test species. Research conducted by this group suggests that this pulsed UV system may offer a useful method for the disinfection of drinking and wastewater supplies.