Kocuria Species Research Articles

Draft genome of Kocuria polaris CMS 76or(T) isolated from cyanobacterial mats, McMurdo Dry Valley, Antarctica: an insight into CspA family of proteins from Kocuria polaris CMS 76or(T).

Kocuria polaris strain CMS 76or(T) is a gram-positive, orange-pigmented bacterium isolated from a cyanobacterial mat sample from a pond located in McMurdo Dry Valley, Antarctica. It is psychrotolerant, orange pigmented, hydrolyses starch and Tween 80 and reduces nitrate. We report the 3.78-Mb genome of K. polaris strain CMS 76or(T), containing 3416 coding sequences, including one each for 5S rRNA, 23S rRNA, 16S rRNA and 47 tRNA genes, and the G+C content of DNA is 72.8%. An investigation of Csp family of proteins from K. polaris strain CMS 76or(T) indicated that it contains three different proteins of CspA (peg.319, peg.2255 and 2832) and the length varied from 67 to 69 amino acids. The three different proteins contain all the signature amino acids and two RNA binding regions that are characteristic of CspA proteins. Further, the CspA from K. polaris strain CMS 76or(T) was different from CspA of four other species of the genus Kocuria, Cryobacterium roopkundense and E. coli indirectly suggesting the role of CspA of K. polaris strain CMS 76or(T) in psychrotolerant growth of the bacterium.

Kocuria dechangensis sp. nov., an actinobacterium isolated from saline and alkaline soils

A Gram-stain positive, strictly aerobic, non-motile and coccus-shaped actinobacterium, designated strain NEAU-ST5-33(T), was isolated from saline and alkaline soils in Dechang Township, Zhaodong City, PR China. It formed beige-yellow colonies and grew at NaCl concentrations of 0-5% (w/v) (optimum 0%), at pH 6.0-9.0 (optimum pH 7.0) and over a temperature range of 4-50 °C (optimum 35 °C). Based on 16S rRNA gene sequence analysis, strain NEAU-ST5-33(T) was phylogenetically closely related to the type strains of species of the genus Kocuria, Kocuria polaris CMS 76or(T), Kocuria rosea DSM 20447(T), Kocuria turfanensis HO-9042(T), Kocuria aegyptia YIM 70003(T), Kocuria himachalensis K07-05(T) and Kocuria flava HO-9041(T), with respective sequence similarities of 98.8%, 98.8%, 98.3%, 98.1%, 98.1% and 97.9%. DNA-DNA hybridization relatedness values of strain NEAU-ST5-33(T) with type strains of the closely related species ranged from 54 ± 1% to 34 ± 1%. The DNA G+C content was 61.2 mol%. The major fatty acids (>5%) were C15 : 0 anteiso, C15 : 0 iso and C16 : 1ω7c and/or C16 : 1ω6c. The major menaquinone detected was MK-8 (H2), and the polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unknown aminolipid and one unknown lipid. On the basis of the genotypic, chemotaxonomic and phenotypic data, we propose that strain NEAU-ST5-33(T) represents a novel species of the genus Kocuria, with the name Kocuria dechangensis sp. nov. The type strain is NEAU-ST5-33(T) ( = CGMCC 1.12187(T) = DSM 25872(T)).

Kocuria Species Peritonitis: Although Rare, we Have to Care

Kocuria species are found in the environment and on human skin. These micro-organisms are generally considered to be nonpathogenic saprophytes, rarely causing infection. However, the peritoneum has been reported to be a site of Kocuria infection. We reviewed all cases of peritonitis in peritoneal dialysis (PD) patients caused by Kocuria species that were reported in the worldwide literature. In total, 12 episodes of Kocuria species peritonitis have been reported in 9 PD patients. The median age of the patients was 62 years (range: 8 - 78 years). In the reported episodes, 4 different Kocuria species were isolated, with K. varians being the predominant species (41.7%). The most common initial symptom was abdominal pain (83.3%), followed by turbid effluent (75%) and fever (33.3%). Intraperitoneal first-generation cephalosporins and glycopeptides were the most-used antibiotics, with first-generation cephalosporins being more often preferred as first-line therapy. The median duration of treatment was 14 days, and in 2 episodes, the Tenckhoff catheter was removed. Although Kocuria peritonitis in PD patients is rare, it should be promptly treated because relapses can occur, especially with K. varians episodes.

Kocuria dacryocystitis infection, caused by kocuria ocularis sp. Nov.

Introduction: Kocuria spp. rarely cause infectious disease but can be opportunistic pathogens in immunocompromised patients. The numbers of documented infections are low but rising. Case presentation: A 74‐year‐old woman presented with acute, painful swelling of the medial canthus of the left eye. The skin surface was red and acutely painful to the touch. Because of several clinical relapses despite antibiotic treatment, surgical dacryocystorhinostomy with marsupialization of the lacrimal sac into the nasal cavity was performed. Lacrimal sac drainage samples were analysed and yielded Kocuria. This is, to our knowledge, the first case of Kocuria dacryocystitis. Furthermore, the dacryocystitis was caused by a novel species of Kocuria, which we suggest should be named Kocuria ocularis. Conclusion: Kocuria related to ocular infection has not yet been documented. This report expands the clinical spectrum of diseases caused by these potentially underestimated pathogens.

Isolation of Cytochrome P-450cam from Alkaliphilic bacteriaKocuria sp. DL

Cytochrome P-450 is a terminal oxidase, involved in biotransformation of endogenous and exogenous substances. Cytochrome P-450cam is isozyme present in various bacteria. CYP-450cam was purified from alkaliphilic bacteria, Kocuria sp. DL, isolated from pristine alkaline Crater Lake, Lonar, (Maharashtra State), India. Identification of this bacterial strain was carried out by analysis of 16S rRNA gene sequence (Kocuria species DL strain). The content of CYP-450cam was found to be 2.68 n mol/mg of protein in 50 h grown culture under aerobic condition. Purification of CYP-450cam was carried out by applying analytical procedures at 4 oC. The Km values for both aminopyrine and acetanilide were 0.4 and 0.25 mM, respectively. The maximal velocity Vmax was 12.98 and 13.2 mM/mg of protein per min for aminopyrine and acetanilide, respectively. The optimal pH and temperatures were found to be 7.4 and 40 oC, respectively. The purification fold was found to be 6. The molecular mass was found to be 48 kDa. Thermal stability of purified CYP-450cam is up to 40 oC. This enzyme has shown the characteristic type-I and type-II substrate binding spectra.

Biodegradation of the herbicide propanil, and its 3,4-dichloroaniline by-product in a continuously operated biofilm reactor

The persistence of propanil in soil and aquatic environments along with the possible accumulation of toxic degradation products, such as chloroanilines, is of environmental concern. In this work, a continuous small-scale bioprocess to degrade the herbicide propanil, its main catabolic by-product, 3,4-dichloroaniline (3,4-DCA), and the herbicide adjuvants is carried out. A microbial consortium, constituted by nine bacterial genera, was selected. The isolated strains, identified by amplification and sequencing of their 16S rDNA, were: Acidovorax sp., Luteibacter (rhizovicinus), Xanthomonas sp., Flavobacterium sp., Variovorax sp., Acinetobacter (calcoaceticus), Pseudomonas sp., Rhodococcus sp., and Kocuria sp. The ability of the microbial consortium to degrade the herbicide was evaluated in a biofilm reactor at propanil loading rates ranging from 1.9 to 36.8 mg L(-1) h(-1). Complete removal of propanil, 3,4-DCA, chemical oxygen demand and total organic carbon was obtained at propanil loading rates up to 24.9 mg L(-1) h(-1). At higher loading rates, the removal efficiencies decayed. Four of the identified strains could grow individually in propanil, and 3,4-DCA: Pseudomonas sp., Acinetobacter calcoaceticus, Rhodococcus sp., and Xanthomonas sp. The Kokuria strain grew on 3,4-DCA, but not on propanil. The first three bacteria have been related to biodegradation of phenyl urea herbicides or chlorinated anilines. Although some strains of the genera Xanthomonas and Kocuria have a role in the biodegradation of several xenobiotic compounds, as far as we know, there are no reports about degradation of propanil by Xanthomonas or 3,4-DCA by Kocuria species.

Study of the Micrococcaceae and Staphylococcaceae throughout the Manufacture of Dry-Cured Lacón (a Spanish Traditional Meat Product) Made without or with Additives

Micrococcaceae and Staphylococcaceae were enumerated (on SPC agar + 7.5% NaCl) in samples from the surface and the interior of pieces of dry-cured lacon (a Spanish traditional meat product), at different stages of the manufacturing process, and from six different batches (three made without and three with additives -glucose, sodium nitrite, sodium nitrate, sodium ascorbate, and sodium citrate-). The use of additives did not affect the counts or evolution of this microbial group. For four batches (two without and two with additives), a total of 335 strains were isolated and identified by classical methods. Staphylococcus xylosus was the most abundant and constant species throughout manufacture of the batches made without and with additives. Other species of staphylococci were isolated, including: Staph. equorum, Staph. sciuri, Staph. gallinarum, Staph. cohnii, Staph. intermedius, Staph. capitis, Staph. epidermidis, Staph. simulans and Staph. warneri. Species of Micrococcus or Kocuria isolated in very low proportions, included Kocuria varians, K. rosea, Micrococcus lylae and M. luteus.

Kocuria sediminis sp. nov., isolated from a marine sediment sample

A Gram-positive, pinkish-orange pigmented, coccoid strain, FCS-11(T) was isolated from a marine sediment sample taken from Kochi fort area, Kerala, India and subjected to polyphasic taxonomic study. The 16S rRNA gene sequence of the strain was determined and the results of 16S rRNA gene sequence analysis showed that the strain FCS-11(T) should be assigned to the genus Kocuria. The chemotaxonomic data supported this taxonomic placement i.e. menaquinones MK-7(H(2)), MK-8(H(2)) and MK-9(H(2)); major fatty acids anteiso C15:0 and iso-C15:0 and phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) as major polar lipids. Further phylogenetic analysis of the 16S rRNA gene sequence confirmed that the strain FCS-11(T) belonged to the genus Kocuria and is closely related to Kocuria turfanensis MTCC 10790(T) (99.4%) followed by Kocuria polaris MTCC 3702(T) (98.2%), Kocuria rosea MTCC 2522(T) (98.2%), Kocuria flava MTCC 10971(T) (98.2%), Kocuria aegyptia MTCC 10791(T) (98.0%), Kocuria himachalensis MTCC 7020(T) (97.5%) and Kocuria atrinae MTCC 10972(T) (97.1%). However, the DNA-DNA hybridisation values obtained between strain FCS-11(T) and other related strains were well below the threshold that is required for the proposal of a novel species. The G+C content of the genomic DNA was 60.7mol%. The phenotypic and genotypic data showed that the strain FCS-11(T) merits the recognition as a representative of a novel species of the genus Kocuria. It is proposed that the isolate should be classified in the genus Kocuria as a novel species, Kocuria sediminis sp. nov. The type strain is FCS-11(T) (=MTCC 10969(T)=JCM 17929(T)).

Catheter-related bacteraemia and infective endocarditis caused by Kocuria species

We describe five patients with positive blood culture for Kocuria species. Three patients had catheter-related bacteraemia and one had infective endocarditis caused by Kocuria kristinae, and one had a K. marina isolate, which was considered to be a contaminant. Identification of the isolates was further confirmed by 16S rRNA gene sequence analysis. In conclusion, Kocuria species are an unusual cause of infection in immunocompromised patients. Accurate identification with molecular methods is imperative for the diagnosis of these unusual pathogens.

Central venous catheter-related bacteremia caused by Kocuria kristinae: Case report and review of the literature

Kocuria species are unusual human pathogens isolated most commonly from immunocompromised hosts, such as transplant recipients and cancer patients undergoing chemotherapy, or from patients with chronic medical conditions. A case of catheter-related bacteremia with pulmonary septic emboli in a pregnant adult female without chronic medical conditions is described. A review of other reported Kocuria infections is provided.

Isolation of two Kocuria species capable of growing on various polycyclic aromatic hydrocarbons

Different samples collected from crude oil contaminated beach were enriched for isolation of bacterial strains capable of growing on naphthalene, phenanthrene and fluoranthene. Respiratory reduction of WST-1{4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate} to a colored formazan showed that one isolated strain CMG2028, identified as Kocuria flava by 16s rRNA, grew on naphthalene and phenanthrene while CMG2042, identified as Kocuria rosea grew on all three polycyclic aromatic hydrocarbons (PAHs). In naphthalene degradation test, 64 and 47% residual naphthalene was extracted after ten days of incubation from culture medium of K. rosea CMG2042 and K. flava CMG2028, respectively, when provided with 0.5 mg ml-1 concentration as sole carbon source. Due to addition of 0.5 mg ml-1 yeast extract as additional carbon source, residual naphthalene extracted was 41 and 55% from K. rosea CMG2042 and K. flava CMG2028, respectively. Both strains exhibited growth on 0.01 mg ml-1 phenanthrene and fluoranthene in yeast extract added or omitted medium but only K. rosea CMG2042 degraded 9% phenanthrene as a sole carbon source. Both strains had growth on minimal agar plates coated with Iranian light crude oil as sole carbon source and on agar medium added with yeast extract.

Degradation of pentachlorophenol by Kocuria sp. CL2 isolated from secondary sludge of pulp and paper mill

A pentachlorophenol (PCP) degrading bacterium was isolated and characterized from sludge of pulp and paper mill. This isolate used PCP as its sole source of carbon and energy and was capable of degrading this compound, as indicated by stoichiometric release of chloride and biomass formation. Based on morphology, biochemical tests, and 16S rRNA gene sequence analysis this strain was identified as Kocuria sp. CL2. High Performance Liquid Chromatography (HPLC) analysis revealed that this strain was able to degrade PCP up to a concentration of 600mg/l. This is first time we are reporting the degradation of PCP by the Kocuria species. This isolate was also able to remove 58.64% of PCP from the sludge within two weeks. This study showed that the removal efficiency of PCP by CL2 was found to be very effective and can be used in degradation of PCP containing pulp paper mill waste in the environment.

Kocuria salsicia sp. nov., isolated from salt-fermented seafood

Strain 104(T) was isolated from a traditional salt-fermented seafood in Korea. It was a Gram-positive, non-motile, coccus-shaped bacterium. It formed lemon-yellow, opaque colonies that were circular with entire margins. Optimal growth occurred at 30-37 °C, pH 7-8 and in the presence of 0-2 % (w/v) NaCl. Phylogenetic analysis of 16S rRNA gene sequences from strain 104(T) and reference species of the genus Kocuria indicated that strain 104(T) formed an independent line. The G+C content of the chromosomal DNA was 60.6 mol%. MK-7 was the major menaquinone and the predominant fatty acids were anteiso-C(15 : 0) (76.7 %), anteiso-C(17 : 0) (10.9 %) and iso-C(16 : 0) (4.5 %). Strain 104(T) was most closely related to Kocuria rhizophila TA68(T) (98.9 % 16S rRNA gene sequence similarity). The DNA-DNA hybridization value between strain 104(T) and K. rhizophila TA68(T) was 14.1±3.4 %. On the basis of this polyphasic taxonomic analysis, strain 104(T) appears to represent a novel species in the genus Kocuria. The name Kocuria salsicia sp. nov. is proposed. The type strain is 104(T) (=KACC 21128(T)=JCM 16361(T)).

Kocuria atrinae sp. nov., isolated from traditional Korean fermented seafood

A novel actinobacterium, strain P30(T), was isolated from jeotgal, a traditional Korean fermented seafood. Cells were aerobic, Gram-positive, non-motile and coccoid. Optimal growth occurred at 30-37 degrees C, at pH 8-9 and in the presence of 0-2 % (w/v) NaCl. Based on 16S rRNA gene sequence analysis, strain P30(T) was phylogenetically closely related to Kocuria carniphila, Kocuria gwangalliensis, Kocuria rhizophila, Kocuria marina, Kocuria rosea and K. varians with levels of similarity of 98.6, 98.2, 98.1, 97.4, 97.3 and 97.3 %, respectively, to the type strains of these species. Levels of DNA-DNA relatedness between strain P30(T) and the type strains of K. carniphila, K. rhizophila, K. marina, K. rosea and K. varians were 37, 43, 37, 25 and 17 %, respectively. The predominant menaquinone of strain P30(T) was MK-7. Major cellular fatty acids were anteiso-C(15 : 0), iso-C(15 : 0) and iso-C(16 : 0). The genomic DNA G+C content of strain P30(T) was 70.2 mol%. Based on these data, strain P30(T) is considered to represent a novel species of the genus Kocuria, for which the name Kocuria atrinae sp. nov. is proposed. The type strain is P30(T) (=KCTC 19594(T)=JCM 15914(T)).

Kocuria koreensis sp. nov., isolated from fermented seafood

A Gram-positive, aerobic, non-motile and coccoid actinobacterium, designated P31(T), was isolated from a traditional, fermented seafood. The strain was catalase-positive and oxidase-negative. Cells grew in the presence of 0-15.0 % (w/v) NaCl, and at pH 5-10 and 15-37 degrees C. Major cellular fatty acids were anteiso-C(15 : 0), anteiso-C(17 : 0) and iso-C(16 : 0). Strain P31(T) contained MK-7 as the predominant menaquinone. The DNA G+C content of the genomic DNA of strain P31(T) was 65.2 mol%. A phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain P31(T) was most closely related to Kocuria kristinae DSM 20032(T), with 96.9 % similarity, and these two strains clustered together in constructed phylogenetic trees. The DNA-DNA hybridization value between strain P31(T) and K. kristinae DSM 20032(T) was 21.1 %. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, it is suggested that strain P31(T) represents a novel species of the genus Kocuria, for which the name Kocuria koreensis sp. nov. is proposed. The type strain is P31(T) (=KCTC 19595(T)=JCM 15915(T)).

Kocuria gwangalliensis sp. nov., an actinobacterium isolated from seawater

A novel aerobic, non-motile, Gram-positive, pink-orange-pigmented, coccoid marine bacterium, designated strain SJ2(T), was isolated from the Gwangalli coast of Korea. The taxonomic position of strain SJ2(T) was determined based on 16S rRNA gene sequence analysis, fatty acid patterns and physiological reaction profiles. The full-length 16S rRNA gene sequence of strain SJ2(T) showed highest similarity to those of the type strains of Kocuria carniphila and Kocuria marina. Strain SJ2(T) exhibited mean levels of DNA-DNA relatedness of 17 and 35 % to the type strains of K. carniphila and K. marina, respectively. Based on these results, strain SJ2(T) is considered to represent a novel species of the genus Kocuria, for which the name Kocuria gwangalliensis sp. nov. is proposed. The type strain is SJ2(T) (=KCCM 42914(T) =LMG 24672(T)).

Kocuria halotolerans sp. nov., an actinobacterium isolated from a saline soil in China

A Gram-positive actinobacterium, designated strain YIM 90716(T), was isolated from a saline soil sample collected from Ganjiahu Suosuo Forest National Nature Reserve in Xinjiang Province, north-west China. The new isolate contained lysine, glutamic acid and alanine with peptidoglycan type Lys-Ala(3) (variation A3alpha). The major phospholipids were phosphatidylglycerol and diphosphatidylglycerol. The predominant menaqinone was MK-7(H(2)). The major fatty acids were anteiso-C(15 : 0), iso-C(16 : 0) and anteiso-C(17 : 0). The DNA G+C content of strain YIM 90716(T) was 68.0 mol%. Chemotaxonomic properties supported the affiliation of strain YIM 90716(T) to the genus Kocuria. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the organism was related most closely to Kocuria kristinae DSM 20032(T) (96.8 % similarity) and showed lower levels of 16S rRNA gene similarity (<96.5 %) with the type strains of other species of the genus Kocuria. The results of fatty acid analysis and physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain YIM 90716(T) from its closest relatives. On the basis of data from the present polyphasic study, strain YIM 90716(T) is considered to represent a novel species of the genus Kocuria, for which the name Kocuria halotolerans sp. nov. is proposed. The type strain is YIM 90716(T) (=DSM 18442(T)=KCTC 19172(T)=CCTCC AB 206069(T)).

Kocuria flava sp. nov. and Kocuria turfanensis sp. nov., airborne actinobacteria isolated from Xinjiang, China

Two coloured bacteria were isolated from the same plate for detecting cultivable bacteria from the air of Xinjiang in China. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the isolates were members of the genus Kocuria, in which they represented two novel lineages. Although the two strains presented high 16S rRNA gene sequence similarity (above 97 %), their DNA G+C contents were very different (6 mol%). The G+C contents of strains HO-9041(T) and HO-9042(T) are 71 and 65 mol%, respectively. DNA relatedness analysis and other taxonomic evidence supports the placement of the two isolates in the genus Kocuria. The diagnostic diamino acid of the cell-wall peptidoglycan is l-lysine and both strains contain MK-8(H(2)) and MK-9(H(2)) as major menaquinones. In addition, they share similar fatty acid patterns containing straight-chain saturated and iso- and anteiso-branched acids, with a major component being anteiso-C(15:O). Genotypic, morphological and physiological characteristics are used to describe two novel species of Kocuria, for which the names Kocuria flava sp. nov. (type strain HO-9041(T) =CCTCC AB 206,106(T) =KCTC 19,306(T)) and Kocuria turfanensis sp. nov. (type strain HO-9,042(T) =CCTCC AB 206,107(T) =KCTC 19,307(T)) are proposed.

Metabolic Activity and Phylogenetic Diversity of Reed (Phragmites australis) Periphyton Bacterial Communities in a Hungarian Shallow Soda Lake

In the present study, the species composition and potential metabolic activities of bacterial communities of reed Phragmites australis (Cav.) (Trin. ex Steudel) periphyton from Lake Velencei were studied by cultivation-based and metabolic fingerprinting methods. Serially diluted spring biofilm samples were used to test the community-level physiological profiling (CLPP) using BIOLOG microplates, and for plating onto different media. On the basis of their morphological, biochemical, and physiological test results, 173 strains were clustered by numerical analysis. Representatives of amplified ribosomal DNA restriction analysis (ARDRA) groups were identified by their 16S rDNA sequence comparison. Based on the results of the CLPP investigations, regional differences were detected among the utilized substrate numbers and types, parallel with the increase in incubation time. The phenotypic test results of the strains showed considerable variability with respect to the sampling sites and the media used for cultivation. The most frequently isolated strains were identified as members of genera Agrobacterium, Pseudomonas (P. anguilliseptica, P. marginalis, P. alcaligenes, P. fragi) with aerobic or facultative anaerobic respiratory metabolism, and the species Aeromonas sobria and A. veronii with strong facultative fermentative metabolism. Other strains were identified as Gram-positive Arthrobacter, Bacillus, and Kocuria species. The rarely isolated strains were members of beta-Proteobacteria (Acidovorax, Delftia, Hydrogenophaga, and Rhodoferax), gamma-Proteobacteria (Psychrobacter and Shewanella), low G + C Gram-positives (Brevibacillus, Paenibacillus, and Exiguobacterium) and high G + C Gram-positives (Aureobacterium and Microbacterium).

Kocuria himachalensis sp. nov., an actinobacterium isolated from the Indian Himalayas

A reddish orange bacterium, strain K07-05(T), was isolated from soil during a study of the bacterial diversity of a cold desert of the Indian Himalayas and was studied by using a polyphasic approach. The organism had morphological and chemotaxonomic properties consistent with its classification in the genus Kocuria. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain K07-05(T) was closely related to Kocuria rosea DSM 20447(T) and Kocuria polaris MTCC 3702(T) (98.1 and 97.8 % sequence similarity, respectively), whereas the sequence similarity values with respect to the other Kocuria species with validly published names were between 96.4 and 94.2 %. However, the genomic relatedness, as shown by DNA-DNA hybridization, of strain K07-05(T) and K. polaris MTCC 3702(T) is 49.5 % and that with K. rosea MTCC 2522(T) is 24.0 %. The DNA G+C content of the strain is 75.3 mol%. The above data in combination with the phenotypic distinctiveness of K07-05(T) clearly indicate that the strain represents a novel species, for which the name Kocuria himachalensis sp. nov. is proposed. The type strain is K07-05(T) (=MTCC 7020(T)=DSM 44905(T)=JCM 13326(T)).