Susceptibility ofBacteroides non-fragilis and fusobacteria to amoxicillin,amoxicillin/clavulanate,ticarcillin, ticarcillin/clavulanate,cefoxitin, imipenem and metronidazole

The susceptibility of 234Bacteroides non-fragilis strains and 56 fusobacteria from 12 European centers to amoxicillin, amoxicillin/clavulanate, ticarcillin, ticarcillin/clavulanate, cefoxitin, imipenem and metronidazole was tested and related to -lactamase production. Beta-lactamase production was detected in 42.3 % of theBacteroides strains and 26.8 % of the fusobacteria. The MIC90 of amoxicillin for -lactamase-negative strains was 0.5 µg/ml and the MIC90 of ticarcillin 2.0 µg/ml. In the case of -lactamase-positive strains the MIC90 of amoxicillin (32 µg/ml) and ticarcillin (16 µg/ml) dropped to 1.0 µg/ml upon addition of clavulanate; 65.8 % of these strains were susceptible to amoxicillin and 98.2 % to ticarcillin, but all were susceptible when clavulanate was added. All strains were susceptible to imipenem and metronidazole, and 99.3 % to cefoxitin.     

Beta-lactamase production and susceptibility of US and European anaerobic gram-negative bacilli to beta-lactams and other agents

The susceptibility of 1,476 US and European strains of anaerobic gram-negative bacilli to amoxicillin, amoxicillin/clavulanate, ticarcillin, ticarcillin/clavulanate, cefoxitin, imipenem and metronidazole was determined. All of theBacteroides fragilis group and 51 % of the non-Bacteroides fragilis group were -lactamase positive. Amongst the non-Bacteroides fragilis group, -lactamase positivity rates were higher for US strains (58 %) than for European strains (39 %). All strains were susceptible to imipenem and metronidazole. MIC90s of amoxicillin and ticarcillin for all -lactamase negative strains were 0.5 and 2 µg/ml, respectively. The addition of clavulanate reduced the MIC90s of amoxicillin ( 256 µg/ml) and ticarcillin ( 64 µg/ml) to 16 and 8 µg/ml, respectively, for theBacteroides fragilis group, and to 4 µg/ml for both agents for the non-Bacteroides fragilis -lactamase producing group. Twenty-nine cefoxitin-resistant strains were found, mainly in theBacteroides fragilis group, while 95 -lactamase producing strains (predominantlyBacteroides fragilis group and fusobacteria) did not show synergy between -lactams and clavulanate. Of the newer agents tested, meropenem and piperacillin-tazobactam were the most active (100 % of strains susceptible), followed by amoxicillin-BRL 42715 (99 % of strains susceptible); 94 to 98 % of the strains were susceptible to cefoperazone-sulbactam, tosufloxacin, tempafloxacin and clindamycin. Only 73 % of the strains were susceptible to cefotetan, compared to 91 % to cefoxitin; 88 % of the strains were susceptible to trospectomycin. Overall, all of the -lactam/-lactamase inhibitor combinations, imipenem, meropenem, cefoxitin, tosufloxacin, temafloxacin and clindamycin had good activity against -lactamase producing strains, while all agents tested had good activity against -lactamase negative strains.     

Comparative in vitro activity of cefdinir (CI-983; FK-482) against staphylococci,gram-negative bacilli and respiratory tract pathogens

The in vitro activity of cefdinir (CI-983; FK-482), a new oral cephalosporin, was compared with that of other antimicrobial agents against clinical isolates of staphylocci, gram-negative bacilli and common respiratory tract pathogens. Cefdinir (MIC90 2.0 µg/ml) was more active than cefixime (MIC90 >64 µg/ml) and equally as active as cefuroxime (MIC90 2.0 µg/ml) against oxacillin-susceptible staphylococci. Cefdinir was active againstHaemophilus influenzae, including -lactamase producers (MIC90 0.5 µg/ml),Moraxella catarrhalis (MIC90 0.12 µg/ml),Streptococcus pneumoniae (MIC90 0.06 µg/ml) andStreptococcus pyogenes (MIC90 0.06 µg/ml). The activity of cefdinir against gram-negative bacilli was variable; organisms with chromosomal cephalosporinases were often resistant.     

Multicentre comparative study on the antibacterial activity of FK-037, a new parenteral cephalosporin

The in vitro antibacterial activity of FK-037, a new parenteral cephalosporin structurally related to cefpirome and cefepime, was compared with that of cefotaxime, ceftazidime, aztreonam, cefpirome, cefepime, imipenem and meropenem against 1,837 clinical isolates obtained from three Spanish hospitals. FK-037 inhibited 90 % ofEnterobacteriaceae isolates at 0.25 µg/ml, with the exception ofEnterobacter aerogenes (MIC90 1 µg/ml),Enterobacter cloacae andCitrobacter freundii (MIC90 8 µg/ml). In cefotaxime-and ceftazidime-resistantKlebsiella pneumoniae strains producing SHV-2 and SHV-6 -lactamases, the activity of FK-037, cefpirome and cefepime was similar (MIC range 0.25–32 µg/ml). InEnterobacteriaceae strains hyperproducing chromosomally inducible -lactamases, FK-037 (MIC90 range, 0.25–8 µg/ml) was 8- to 16-fold more active than cefotaxime and ceftazidime but two- to eightfold less active than cefpirome and cefepime. FK-037 and cefpirome were twofold more active than ceftazidime and cefepime againstPseudomonas aeruginosa isolates, with MIC90 values of 16 µg/ml. The activity of FK-037, cefpirome and cefepime was two- to eightfold lower in ceftazidime-resistant derepressedPseudomonas aeruginosa mutants. FK-037 (MIC range, 0.12–2 µg/ml) and the other -lactam agents tested were active against methicillin-susceptible staphylococci; however, only cefpirome and, particularly, FK-037 (MIC90 of 32 µg/ml) displayed some activity against methicillin-resistant strains. In penicillin-susceptible,-intermediate and -resistantStreptococcus pneumoniae isolates, the MIC90s of FK-037 were 0.03, 0.5 and 1 µg/ml, respectively. The corresponding values forStreptococcus viridans isolates were 0.12, 1 and 8 µg/ml, respectively. In bothStreptococcus pneumoniae andStreptococcus viridans isolates, FK-037 displayed activity similar to that of cefotaxime and cefpirome and slightly higher than that of cefepime.     

Activity of beta-lactamase inhibitor combinations onEscherichia coli isolates exhibiting various patterns of resistance to beta-lactam agents

The efficacy of the clinically available -lactam/-lactamase inhibitor combinations (amoxicillin/clavulanic acid (CA), ticarcillin/CA, amoxicillin/sulbactam, and piperacillin/ tazobactam) was evaluated on 300 amoxicillin-resistantEscherichia coli isolates having the main patterns of -lactam resistance. The patterns, which reflect the production of various -lactamase enzymes, were analyzed by a principal component analysis of susceptibility to 11 -lactam antibiotics or -lactam/-lactamase inhibitor combinations. Sixty-two percent of strains were not very susceptible to penicillins, cephalothin, or any -lactam/-lactamase inhibitor combinations except for piperacillin/tazobactam; these strains may represent high-level broad-spectrum -lactamase (so-called penicillinase) production phenotype or inhibitor-resistant TEM-like enzyme production phenotype. Of the strains, 14.7 % were resistant to amoxicillin and ticarcillin compatible with low-level broad-spectrum -lactamase production phenotype; 5.7 % were cefoxitin resistant and were postulated to present a high-level cephalosporinase production phenotype; and 2.6 % were resistant to cephalothin only, attributable to a low-level cephalosporinase production phenotype. Three percent of strains were intermediate or resistant to cefotaxime and may produce an extended-spectrum -lactamase, and the remaining strains (12 %), resistant to all tested antibiotics except for cefotaxime and piperacillin/tazobactam, were hypothesized to produce both broad-spectrum -lactamase plus cephalosporinase. The minimal inhibitory concentration (MIC) for these phenotype patterns indicated that combinations of CA plus amoxicillin or ticarcillin, or sulbactam plus amoxicillin, restored the activity of penicillins against phenotype 1 strains, whereas these combinations remained inactive against the other phenotype strains. Piperacillin plus tazobactam showed the best in vitro effect against the strains of all resistance phenotypes.     

In vitro antibacterial activity and beta-lactamase stability of the new carbapenem SM-7338

The in vitro activity of the new carbapenem SM-7338 was tested in comparison with imipenem, ceftazidime, cefotaxime, flomoxef, cefuzonam and cefmetazole against 2850 clinical bacterial isolates. SM-7338 showed good activity against a broad spectrum of grampositive and gram-negative bacteria. SM-7338 was very active against gram-negative bacteria, inhibiting allEnterobacteriaceae, except 25 % ofSerratia marcescens isolates, at a concentration of 0.78 mg/l. SM-7338 inhibited the majority ofPseudomonas spp. at concentrations of 3.13 mg/l, its activity being twofold higher than that of imipenem. However, the activity of SM-7338 against gram-positive cocci was about one-fourth that of imipenem. Against anaerobes, SM-7338 also had the best activity of the -lactams tested. The compound was inactive against methicillin-resistant staphylococci,Enterococcus faecium, Xanthomonas maltophilia andFlavobacterium spp., as were the other -lactams. SM-7338 was quite stable in the presence of various types of -lactamase, but was hydrolyzed byXanthomonas maltophilia -lactamase, as was imipenem. This high degree of stability was responsible for the potent activity of SM-7388 against -lactamase-producing species such asEnterobacter cloacae, Citrobacter freundii andProteus vulgaris. SM-7338 also showed bactericidal activity against clinical isolates at the MIC or at concentrations slightly above the MIC.     

Lack of additive effect between mechanisms of resistance to carbapenems and other beta-lactam agents inPseudomonas aeruginosa

Eighty-nine clinical isolates resistant (n=61) or susceptible (n=28) to imipenem and exhibiting the main patterns of susceptibility to other -lactam agents (wild type pattern, penicillinase pattern, constitutive cephalosporinase pattern) were studied in order to investigate (i) the mechanism of resistance involved and (ii) whether resistance to carbapenems affects the level of resistance to other -lactam agents and, conversely, if resistance to other -lactam agents affects the level of resistance to carbapenems. For this purpose, the presence of OprD protein in the cell wall was detected by Western blot and -lactamase activity by spectrophotometric assay and isoelectric focusing. OprD expression was not detectable in the imipenem-resistant (MIC16 g/ml) strains. It was decreased in half the strains for which MICs of imipenem were 2 to 8 g/ml and was close to a normal level in the most susceptible strains (MIC 1 g/ml), thus demonstrating a direct correlation between the level of susceptibility to imipenem and the level of OprD expression. No imipenemase activity was detected in imipenem-resistant strains. Synergy between imipenem or meropenem and BRL42715 was observed for all of the strains, demonstrating the role of cephalosporinase in carbapenem resistance. Within each pattern of susceptibility, the mean MICs of -lactam agents other than carbapenems were similar, whether the strains were susceptible or resistant to imipenem. Conversely, the mean MICs of imipenem or meropenem for either the imipenem-resistant or the imipenem-susceptible strains were similar, regardless of the susceptibility of these strains to the other -lactam agents. Thus, when several mechanisms of resistance to -lactam agents are present in the same strain ofPseudomonas aeruginosa, there is no additive effect between these mechanisms.     

Clinical significance of beta-lactamase induction and stable derepression in gram-negative rods

Most strains of enterobacteria andPseudomonas aeruginosa produce chromosomally-determined Class I-lactamases. When synthesized copiously these enzymes cause resistance to almost all-lactams, except imipenem and, sometimes, carbenicillin and tenocillin. Elevated-lactamase production arises transiently, via induction, inPseudomonas aeruginosa andEnterobacter, Citrobacter, Morganella, indole-positiveProteus andSerratia spp. when these organisms are exposed to-lactams. Permanent high-level enzyme production arises via mutation, in the stably-derepressed mutants of these species. These mutants arise spontaneously at high frequency (10^–5–10^–8). Most early penicillins and first-generation cephalosporins are strong inducers of Class I enzymes at sub-inhibitory concentrations, as are cefoxitin and imipenem. Consequently their MICs reflect what lability these antibiotics have to inducibly-expressed-lactamase. Except with imipenem this lability usually is so great that the inducible enzyme causes clinical resistance. Although most other newer cephalosporins and ureidopenicillins are labile to the Class I enzymes they induce poorly below the MIC, and their lability is not reflected in resistance unless secondary inducers (e.g. cefoxitin or imipenem) are present. Although the weak inducer activity of these agents helps to maintain their activity against the inducible cells it renders the drugs highly selective for the pre-existing stably-derepressed mutants. Many cases have been reported where stably-derepressed mutants have overrun inducible populations of bacteria in patients undergoing therapy with-lactamase-labile weak inducers such as ureidopenicillin and third-generation cephalosporins.     

A mouse mutant strain highly resistant to methyl β-carboline-3-carboxylate-induced seizures

The convulsant properties of methyl -carboline-3-carboxylate (-CCM) were evaluated in the TaT-fm/Gnc ^Ta+/+Tfm strain carrying the tabby coat color (Ta) and/or the testicular feminization (Tfm) gene. When injected intraperitoneally within a 5–60 mg/kg dose range, -CCM-induced convulsions in less than 25% of the mice, thus providing evidence for a high resistance of this strain, as compared to classical strains of mice. However, this strain responds normally to the convulsant pentylenetetrazol (PTZ), suggesting a specific resistance to -CCM. Both the Ta gene and the TaTfm/Gnc genetic background were involved in the high resistance to -CCM. In addition, concentrations of neurosteroids and benzodiazepine binding, both modulating GABA_A receptor efficacy, have been measured in order to elucidate the biological mechanisms of drug resistance.     

Activity of meropenem against imipenem-resistant bacteria and selection in vitro of carbapenem-resistantEnterobacteriaceae

The activity of meropenem against 106 imipenem-resistant (MIC 8 mg/l) clinical isolates, and the frequency of resistance to meropenem and imipenem among 24Enterobacteriaceae was determined. Both agents selected colonies on agar but 20–80 % were susceptible after one subculture and 72 % of the mutants reverted to susceptibility 1 to 6 months after selection. All isolates and stable mutants were inhibited by > 1 mg/l meropenem, although the MIC of imipenem was 4–16 mg/l. Three of sixXanthomonas maltophilia isolates were susceptible to meropenem (MICs 2–4 mg/l).Pseudomonas aeruginosa lacking outer membrane protein D2 were resistant to meropenem, although isolates with substantially reduced expression of this protein were susceptible. None of the imipenem-resistant gram-positive bacteria were susceptible to meropenem. There was no clear correlation between altered outer membrane protein expression and decreased susceptibility to carbapenems, and there was no apparent involvement of plasmid or chromosomal -lactamase.     

In vitro interaction between the penem FCE 22101 and ceftazidime

FCE 22101, a penem antimicrobial agent, was found to resist hydrolysis by bacterial-lactamases and to have a strong affinity for Type Ia enzymes. Like imipenem, FCE 22101 was shown to be capable of inducing resistance to a wide range of-lactam antibiotics. FCE 22101 antagonized the in vitro activity of ceftazidime against enteric bacilli that commonly produce inducible enzymes. This penem should not be combined with other-lactams for chemotherapeutic purposes.     

Changes in the susceptibility ofBacteroides fragilis group organisms to various antimicrobial agents 1979–1989

The in vitro activity of metronidazole, chloramphenicol, clindamycin and 11 -lactam antibiotics against 135 clinical isolates of theBacteroides fragilis group was compared. In addition, changes in the resistance patterns of members of theBacteroides fragilis group isolated at the Hospital Universitario San Carlos in Madrid, Spain, between 1979 and 1989 were documented. The most active -lactam drugs were imipenem and -lactamase inhibitor combinations. In 1989, however, two strains were found to be resistant to imipenem and to all other -lactam agents tested. There was no emergence of resistance to metronidazole. Chloramphenicol was very effective: only one resistant strain was detected in 1979 and no chloramphenicol-resistant isolates were found during the rest of the study period. An outbreak of clindamycin resistance was noted in 1982, and the first cefoxitin resistant strains were recovered in 1985. The changing patterns of susceptibility of anaerobic bacteria to antimicrobial agents and the emergence ofBacteroides fragilis strains resistant to new -lactam agents suggest that periodic antimicrobial susceptibility tests should be performed in order to guide the selection of antimicrobial agents for therapy.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

Detection of SHV-5 extended-spectrum beta-lactamase inKlebsiella pneumoniae strains isolated in Italy

Thirty-fiveKlebsiella pneumoniae strains isolated during 1993–1994 in intensive care units of a large Italian hospital were examined for the presence of extended-spectrum -lactamases. Five strains showed a high level of simultaneous resistance to -lactam agents, including ceftazidime and aztreonam, conferred by a large (130 kb) self-transferable plasmid (in 4 of 5 strains). Isoelectrofocusing and hybridisation studies suggest that these enzymes can be identified as SHV-5 extended-spectrum -lactamases. Pulsed-field gel electrophoresis analysis showed three different genomic fingerprinting profiles, while plasmid restriction enzyme digestion revealed three different patterns, demonstrating that the diffusion of SHV-5 -lactamase is not the result of a single strain or plasmid dissemination.     

In vitro activity of OPC-17116 compared to other broad-spectrum fluoroquinolones

The in vitro activity of OPC-17116 was compared to that of five similar fluoroquinolones (ciprofloxacin, enoxacin, norfloxacin, ofloxacin and temafloxacin). A total of 700 isolates from recent cases of clinical bacteremia were tested. Fifty additional stock strains with well-characterized resistance mechanisms were also processed. The minimal concentrations inhibiting 90 % of strains (MIC90) ofEnterobacteriaceae species were for OPC-17116 0.015–0.5 µg/ml and for ciprofloxacin 0.015–0.25 µg/ml.Moraxella catarrhalis, Haemophilus influenzae andNeisseria gonorrhoeae were very susceptible to OPC-17116 (MIC90 0.015 µg/ml) thus being fourfold more active than ciprofloxacin. For all -hemolytic streptococci and pneumococci OPC-17116 MICs were 0.5 µg/ml. The most resistant enteric bacilli were among theCitrobacter freundii andProvidencia rettgeri strains (MIC90 0.5 µg/ml).Pseudomonas aeruginosa strains were comparably susceptible to OPC-17116 (MIC90 0.5 µg/ml). Low pH and CO₂ incubation had an adverse effect on OPC-17116 MICs, and resistance development was documented among current clinical isolates of staphylococci, pseudomonas and someEnterobacteriaceae.     

Comparative in vitro activity of the new oxazolidinones DuP 721 and DuP 105 against Staphylococci and Streptococci

The in vitro activity of DuP 721 and DuP 105, two orally active members of the oxazolidinones, was compared with that of glycopeptides and ciprofloxacin against 185 gram-positive isolates. Ninety percent ofStaphylococcus aureus isolates, including penicillin- and methicillin-resistant strains, were inhibited by DuP 721 at 1 µg/ml and by DuP 105 at 16 µg/ml; DuP 721 inhibited 90% of coagulasenegative staphylococci tested at 1 µg/ml. The MIC90 forStreptococcus faecalis was 4 µg/ml with DuP 721 and 16 µg/ml with DuP 105; DuP 721 inhibited 90% of -hemolytic streptococci of group A at 0.5 µg/ml. Similar results on selected strains were obtained by continuously recording the optical density of cultures.     

Differential modulating effects of retinoic acid on interferon antiviral activity

Summary The effect of retinoic acid (RA) on the antiviral activity of interferons (IFNs) and in the U 937 and WISH cells was examined to ascertain whether or not RA could reduce the effectiveness of IFN-induced resistance to viral infection. Our results indicate that in the U 937 cells, RA (0.1–1.0 µM) had neither enhancing nor suppressive effect on the antiviral activity of IFN- or against the Semliki Forest virus (SFV). However, in the WISH cells, RA had different effects on IFNs and . Thus, while RA (0.1–50 µM) invariably suppressed the activity of IFN-, it enhanced the action of IFN- at low dose (0.1–1.0 µM) but became suppressive at higher concentrations ( 10 µM). Furthermore, higher antiviral activity was consistently obtained when RA (0.1–10 µM) was added prior to either IFN- or IFN- comparing to cultures with IFN alone. In addition, direct correlation between antiviral activity and the amplitude of 2–5 oligoadenylate (A) synthetase activity was not observed. These results suggest that modulation of IFN antiviral activity by RA varies with different systems and is dependent on the sequence of treatment.     

Interpretive criteria and quality control for antimicrobial susceptibility tests of levofloxacin

To confirm preliminary interpretive breakpoints for prototype 5 µg levofloxacin disks, 490 strains were tested in vitro using commercially manufactured disks. For in vitro susceptibility testing, 5 µg levofloxacin disks can be used with interpretive criteria of 12 mm for resistant (MIC 8.0 µg/ml) and 16 mm for susceptible (MIC 2.0 µg/ml). Proposed quality control limits for tests of levofloxacin are as follows:Escherichia coli ATCC 25922, zones 29–37 mm or MIC 0.008–0.03 µg/ml;Pseudomonas aeruginosa ATCC 27853, zones 19–26 mm or MIC 0.5–2.0 µg/ml;Staphylococcus aureus ATCC 25923, zones 24–31 mm;Staphylococcus aureus ATCC 29213, MIC 0.06–0.25 µg/ml andEnterococcus faecalis ATCC 29212, MIC 0.25–2.0 µg/ml.     

Investigation of the corrosion resistance of brass in a dipterex solution

Conclusions 1. Brass undergoes preferential corrosion in a Dipterex solution. The solution most strongly attacks brass with an inhomogeneous structure of and phases.2. To reduce the corrosion rate of brass in this medium it is necessary to achieve maximum homogeneity of the structure by heat treatment and prevent the arrangement of the phase as a continuous network along the grain boundaries of the phase, since metal with such a structure is subject to corrosion at a maximum rate.3. The corroding action of the solution increases with Dipterex concentration, and therefore when operating the sprayer the recommended concentration, 2.5%, should be used.Zhdanovskii Technological Equipment Plant. Translated from Meditsinskaya Tekhnika, No. 2, pp. 42–44, March–April, 1971.     

GM1-gangliosidosis: Chromosome 3 assignment of theβ-galactosidase-A gene (β GAL A )

The structural gene (GAL_A) coding for lysosomal -galactosidase- A (EC 3.2.1.23) has been assigned to human chromosome 3 using man-mouse somatic cell hybrids. Human -galactosidase-A was identified in cell hybrids with a species-specific antiserum to human liver -galactosidase-A. The antiserum precipitates -galactosidase-A from human tissues, cultured cells, and cell hybrids, and recognizes cross-reacting material from a patient with G_M1 gangliosidosis. We have analyzed 90 primary man-mouse hybrids derived from 12 separate fusion experiments utilizing cells from 9 individuals. Enzyme segregation analysis excluded all chromosomes for GAL_A assignment except chromosome 3. Concordant segregation of chromosomes and enzymes in 16 cell hybrids demonstrated assignment of GAL_A to chromosome 3; all other chromosomes were excluded. The evidence suggests that G_M1 gangliosidosis is a consequence of mutation at this GAL_A locus on chromosome 3.