Synthesis and potent antibacterial activity against MRSA of some novel 1,2-disubstituted-1H-benzimidazole-N-alkylated-5-carboxamidines

A series of 28 novel 1,2-disubstituted-1H-benzimidazole-N-alkylated-5-carboxamidine derivatives were synthesized and evaluated for in vitro antibacterial activities against Staphylococcus aureus and methicillin resistant S. aureus (MRSA) by the tube dilution method. The results showed that compounds 45-46 and 55-57, having 3,4-dichloro substituted phenyl at the position C-2, of N-bulky alkyl substituted benzimidazolecarboxamidines exhibited the greatest activity with MIC values of 1.56-0.39 microg/ml.     

Synthesis and antibacterial evaluation of novel 4-alkyl substituted phenyl beta-aldehyde ketone derivatives

A series of novel 4-alkylphenyl beta-aldehyde ketones and their derivatives were designed and synthesized on the basis of the chemical structures of Houttuynin and beta-lactam antibiotics. Antibacterial activities of these compounds were investigated. The results demonstrated that most of the compounds tested had moderate antibacterial activities against gram-positive pathogen Staphylococcus aureus (ATTC-25923) than Houttuynin, and gram-positive bacteria were more susceptible to the compounds than gram-negative bacteria. Compound 23 was found to be the most potent compound with MIC of 1.0 microg/mL against S. aureus. Particularly, compounds 16, 22 and 23 showed more active antibacterial activities against the clinically important pathogenic bacteria, methicillin-resistant S. aureus (MRSA) than Houttuynin and levofloxacin. The preliminary structure-activity relationship (SAR) analysis suggested that (1) the introduction of appropriate alkyl substituents into position 4 of phenyl ring enhanced antibacterial activities of these compounds, and isopropyl substituent might be more favorable; (2) the presence of ketone carbonyl moiety might play a vital role in determining significant antibacterial activities of these compounds.     

耐甲氧西林金黄色葡萄球菌的分布及联合药物体外抗菌活性分析

目的了解110株耐甲氧西林金黄色葡萄球菌(MRSA)的分布及联合用药对其体外的抗菌活性,以指导临床治疗MRSA感染合理选择抗菌药物。方法常规方法培养分离细菌,用金黄色葡萄球菌乳胶试剂盒及VITEK-2细菌分析仪鉴定到种,MRSA鉴定应用乳胶凝集试剂盒;利用微量稀释法测定利奈唑胺等5种抗菌药物的物体外抗菌活性,按CLSI规定的标准进行。结果从医院感染的标本中分离获得110株MRSA,ICU分离率为36.4%;110株MRSA对利奈唑胺、万古霉素、替考拉宁均敏感;对左氧氟沙星、利福平耐药率分别为96.3%和71.8%;利福平分别与利奈唑胺、万古霉素、替考拉宁联合FIC<1的协同作用均>74.5%,左氧氟沙星分别与利奈唑胺、万古霉素、替考拉宁联合FIC<1的协同作用均<35.4%;各种联合药物间差异有统计学意义(P<0.01)。结论医院ICU是MRSA感染的重要来源,应对其加强医院感染的监测和控制;利奈唑胺、万古霉素、替考拉宁是治疗MRSA感染的首选药物,对中重度感染患者应参考药敏试验结果及患者的个体情况结合联合药物进行优化治疗。     

Design, synthesis and antibacterial activity of novel actinonin derivatives containing benzimidazole heterocycles

A series of novel actinonin derivatives containing a benzimidazole heterocycle linked as amide isostere have been designed and synthesized. The structures of all the synthesized compounds were confirmed by analytical and spectroscopic methods. All the compounds were evaluated in vitro against Staphylococcus aureus, Klebsiella pneumoniae, and Sarcina lutea. Among them, compound 1a with unsubstituted benzimidazole ring exhibited potent antibacterial activities.     

Epidemiology of capsular and surface polysaccharide in Staphylococcus aureus infections complicated by bacteraemia

Staphylococcus aureus is a leading cause of serious hospital- and community-acquired infections. The discovery of serologically distinct capsular polysaccharides on the surface of clinical isolates has allowed the development of vaccines and passive protective immunity. We have studied patient characteristics, infection characteristics and the surface and capsular polysaccharide serotype distribution in patients with S. aureus infections complicated by bacteraemia admitted to VA hospitals in Maryland between 1995 and 2000. Nine hundred and ninety-three blood cultures from 331 patients were positive for S. aureus. Thirty-eight percent of patients had diabetes, 11% had end-stage renal failure, and 23% were injection drug users. Forty-two percent of infections were caused by methicillin-resistant strains (MRSA), and 60% were acquired during hospitalization. Serotyping of the first available isolate per patient (N=234 isolates) using polyclonal antibodies showed three major phenotypes--42%, type 8 (T8) capsule; 50%, type 5 (T5) capsule; and 8%, 336 polysaccharide. MRSA isolates were significantly more likely to be T5 than methicillin-susceptible isolates (66% vs. 39%, P<0.001). The proportion of T5 MRSA increased significantly (years 1-2: 41%; years 3-4: 65%; years 5-6: 90%, P<0.001). This large sample of patients with serious S. aureus infection confirms that capsular polysaccharides T5 and T8 cause most human infections, and together with serotype 336, account for nearly all those with bacteraemia.     

利奈唑胺联用对耐甲氧西林金黄色葡萄球菌体外抗菌活性的研究

目的 探讨利奈唑胺与3种常用抗菌药物(利福平、左氧氟沙星、庆大霉素)分别联用对临床分离的MRSA的体外抗菌活性,为临床有效地治疗MRSA感染提供理论依据.方法 常规方法培养分离细菌,获得纯培养后用VITEK全自动微生物分析仪鉴定金黄色葡萄球菌,再根据CLSI(2006年)标准,微量肉汤法进行最低抑菌浓度(MIC值)的测定及药敏试验,并计算分级抑菌浓度(FIC)指数.结果 利奈唑胺、左氧氟沙星、利福平、庆大霉素对30株MRSA的MIC50分别为0.5～4、16～256、8～256、128～1024 μg/ml,MIC90分别为4、256、256、1024μg/ml;利奈唑胺与左氧氟沙星联用MIC90降为2μg/ml,利奈唑胺与利福平联用MIC90降为1μg/ml,利奈唑胺与庆大霉素联用MIC90降为2 ug/ml.结论 利奈唑胺对MRSA有较高敏感性,利奈唑胺与利福平联合应用以协同作用为主,利奈唑胺与左氧氟沙星、庆大霉素联合应用以协同和相加作用为主,二者均无拮抗作用.     

Synthesis and in vitro antibacterial activity of 7-(3-alkoxyimino-4-amino-4-methylpiperidin-1-yl) fluoroquinolone derivatives

A series of novel 7-(3-alkoxyimino-4-amino-4-methylpiperidin-1-yl)fluoroquinolone derivatives were designed, synthesized and evaluated for their in vitro antibacterial activity and cytotoxicity. All of the target compounds have potent antibacterial activity against the tested Gram-positive and Gram-negative strains, and exhibit good potency in inhibiting the growth of Staphylococcus aureus including MRSA, Staphylococcus epidermidis including MRSE and Streptococcus pneumoniae (MICs: 0.125-4 μg/mL). Compound 22, with the best activity against Gram-positive strains, is 4-16 fold more potent than gemifloxacin, gatifloxacin and levofloxacin against Enterococcus faecalis, and 16- and 4-fold more potent than levofloxacin against S. epidermidis 09-6 and S. pneumoniae 08-4, respectively.     

2011—2015年四川省金黄色葡萄球菌对万古霉素及利奈唑胺耐药性变迁

目的了解四川省金黄色葡萄球菌对万古霉素、利奈唑胺耐药情况,为临床抗感染治疗提供参考依据。方法收集四川省71所医院2011-2015年临床分离的金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌（MRSA）,并计算每年万古霉素与利奈唑胺对金黄色葡萄球菌、MRSA的最低抑菌浓度（MIC）值。结果5年共收集51 976株金黄色葡萄球菌,包括MRSA 14 361株,MRSA检出率由2011年的36.02%下降至2015年的25.56%,呈下降趋势(χ2=160.72,P＜0.05）。2011-2015年万古霉素对金黄色葡萄球菌的MIC50值分别为：1、0.5、0.5、1、1 μg/mL,MIC90值由1 μg/mL上升至2 μg/mL;利奈唑胺MIC50值均为2 μg/mL,MIC90值由2 μg/mL上升至4 μg/mL。2011-2015年万古霉素对MRSA的MIC50、MIC90值变化较明显,分别由0.5、1 μg/mL上升至2 μg/mL;利奈唑胺MIC50值均为2 μg/mL,MIC90值由2 μg/mL上升至4 μg/mL。结论MRSA的检出率有下降趋势,但万古霉素、利奈唑胺对其MIC50及MIC90值总体有上升趋势,需继续加强细菌耐药监测,为临床合理应用抗菌药物提供帮助。     

万古霉素和利奈唑胺对耐甲氧西林金黄色葡萄球菌防耐药变异浓度的测定

目的检测万古霉素和利奈唑胺对MRSA的抗菌活性及两种药物对其耐药突变株的选择能力。方法按照美国临床实验室标准研究院推荐的琼脂平板二倍稀释法测定万古霉素和利奈唑胺对临床分离的122株MRSA的最低抑菌浓度（MIC）;平板稀释法测定二者对MRSA的防耐药变异浓度（MPC）。结果万古霉素对122株MRSA的MICrang为0.5～4.0μg/ml,MIC50为1.0μg/ml,MIC90为2.0μg/ml,MPC90为22.4μg/ml,选择指数为11.2;利奈唑胺对122株MRSA的MICrang为1.0～4.0μg/ml,MIC50和MIC90均为2.0μg/ml,MPC90为5.6μg/ml;选择指数为2.8。结论万古霉素和利奈唑胺对MRSA的抗菌活性均较好,作用于MRSA均不容易筛选出耐药突变株;利奈唑胺限制MRSA耐药突变菌株的选择能力强于万古霉素,较万古霉素更不容易筛选出耐药突变株。     

Antibacterial effects of plant-derived extracts on methicillin-resistant Staphylococcus aureus

Natural chemicals have been reported to have antibacterial effects against a variety of bacteria. The present study evaluated the antibacterial effects of commercially available grape-seed extract (GSE), pomegranate polyphenols (PP), and lab-prepared cranberry proanthocyanidins (C-PAC) against two strains of methicillin-resistant Staphylococcus aureus (MRSA). GSE, PP, and C-PAC at concentrations of 2 mg/mL, 10 mg/mL, or controls were mixed with equal volumes of overnight cultures of MRSA at ~6 log(10) colony-forming units (CFU)/mL and incubated for 0, 1, 2, 8, and 24 h at 37°C. Treatments were neutralized/stopped using tryptic soy broth containing 3% beef extract. Serial dilutions of the treated MRSA strains and controls were spread-plated on trypticase soy agar and incubated for 24-48 h at 37°C and colonies were counted. Among the three tested agents, GSE at 1 and 5 mg/mL was found to be most effective against MRSA, resulting in a 2.9-4.0 log(10) CFU/mL reduction of both strains after 2 h at 37°C. PP at 1 and 5 mg/mL was found to cause 1.1-2.3 log(10) CFU/mL reduction, while C-PAC at 1 mg/mL caused <1 log(10) CFU/mL reduction of the two MRSA strains after 2 h at 37°C. All three extracts at the tested concentrations decreased the two MRSA strains to undetectable levels within 24 h, with the exception of 1 mg/mL PP for strain 33591. Scanning electron microscopy of MRSA after 2 h of treatment showed that GSE and PP caused bacterial cell wall alteration, with negligible effect observed by C-PAC treatment. However, the in vivo activity and clinical safety applications of GSE, PP, and C-PAC need to be evaluated before suggestion for use as a treatment/control measure.     

Antimicrobial activity of berberine alone and in combination with ampicillin or oxacillin against methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) bacteria have been responsible for substantial morbidity and mortality in hospitals because they usually have multidrug resistance. Some natural products are candidates as new antibiotic substances. In the present study, we investigated the antimicrobial activity of berberine, the main antibacterial substance of Coptidis rhizoma (Coptis chinensis Franch) and Phellodendri cortex (Phellodendron amurense Ruprecht), against clinical isolates of MRSA, and the effects of berberine on the adhesion to MRSA and intracellular invasion into human gingival fibroblasts (HGFs). Berberine showed antimicrobial activity against all tested strains of MRSA. Minimum inhibition concentrations (MICs) of berberine against MRSA ranged from 32 to 128 microg/mL. Ninety percent inhibition of MRSA was obtained with 64 microg/mL or less of berberine. In the checkerboard dilution test, berberine markedly lowered the MICs of ampicillin and oxacillin against MRSA. An additive effect was found between berberine and ampicillin, and a synergistic effect was found between berberine and oxacillin against MRSA. In the presence of 1-50 microg/mL berberine, MRSA adhesion and intracellular invasion were notably decreased compared with the vehicle-treated control group. These results suggest that berberine may have antimicrobial activity and the potential to restore the effectiveness of beta-lactam antibiotics against MRSA, and inhibit the MRSA adhesion and intracellular invasion in HGFs.     

Synthesis, characterization and in vitro antibacterial activity of new steroidal thiazolo quinoxalines

Herein, we report the synthesis of different steroidal thiazolo quinoxaline derivatives as antibacterial agents against Escherichia coli. Steroidal ketone thiosemicarbazones (4-6) were obtained from corresponding ketones (1-3) by refluxing with thiosemicarbazide. The thiosemicarbazones on reaction with 2,3-dichloroquinoxalines at 80 degrees C gives 3beta-acetoxy-5alpha-cholestan-6-[thiazolo(4,5-b)quinoxaline-2-yl-hydrazone] (7), 3beta-chloro-cholestan-6-[thiazolo(4,5-b)quinoxaline-2-yl-hydrazone] (8), and 5alpha-cholestan-6-[thiazolo(4,5-b)quinoxaline-2-yl-hydrazone] (9). The structures of the compounds were evident by elemental, IR, (1)H NMR and FAB mass spectral analyses. The antibacterial activities of these compounds were evaluated by disk diffusion method against the culture of E. coli and the results were compared with the standard drug amoxicillin. The results reveal that the compounds are better antibacterial agents as compared to amoxicillin. Among all the three compounds (7-9), compound 8 showed better zone of inhibition.     

Synthesis, antimicrobial activity and possible mechanism of action of 9-bromo-substituted indolizinoquinoline-5,12-dione derivatives

A series of 9-bromo-substituted indolizinoquinoline-5,12-dione derivatives was synthesized. Antimicrobial activity assessment indicates that compounds 1, 26, 27 and 28 exhibit strong activity against gram-positive bacterial strains, including Beta-hemolytic streptococcus CMCC32210, Staphylococcus aureus ATCC25923, Staphylococcus epidermidis ATCC12228, Enterococcus faecalis ATCC29212 and methicillin-resistant S. aureus ATCC43300 (MRSA). Compound 27 shows the best anti-MRSA activity with an MIC value of 0.031 μg/ml. To assess the mechanism of action, the inhibitory activities of compound 1 against DNA gyrase and DNA topoisomerase IV were also measured. The results indicate that compound 1 has strong inhibitory effects on the Escherichia coli DNA gyrase supercoiling activity and S. aureus Topo IV relaxing activity.     

Surveillance for mupirocin resistance following introduction of routine peri-operative prophylaxis with nasal mupirocin

The authors have previously described the successful use of a five-day peri-operative prophylaxis regimen using nasal mupirocin and topical triclosan (PPNMTT) to prevent methicillin-resistant Staphylococcus aureus (MRSA) infection. The present article describes the results of repeated point-prevalence surveillance for four years to determine whether mupirocin resistance has emerged in surgical units using empirical, short-term, peri-operative prophylaxis with nasal mupirocin. Before starting PPNMTT and every six months thereafter for four years, point-prevalence surveillance was performed for nasal S. aureus carriage in all patients on five orthopaedic surgery wards, one vascular surgery ward and one elderly medicine control ward. S. aureus screening and clinical isolates (surgical patients) were undertaken for low- [minimum inhibitory concentration (MIC) 8-128 mg/L] and high-level (MIC > 128 mg/L) mupirocin resistance. All isolates were phage typed to determine whether there was evidence of the spread of clonal mupirocin-resistant strains. Of 593, 139 and 206 nasal screening swabs (taken after the regimen had started) from orthopaedic, vascular and control patients, 28%, 24% and 48% (orthopaedic/vascular surgery vs elderly medicine, P < 0.001) yielded S. aureus isolates, respectively, and 12%, 11% and 30% (P < 0.001) were MRSA positive, respectively. Of the S. aureus nasal screen isolates from orthopaedic/vascular surgery and control patients, 5% and 4%, respectively, were low-level mupirocin resistant (P > 0.1). Of 286 (orthopaedic/vascular surgery) and 68 (elderly medicine) clinical S. aureus isolates obtained after the regimen had started, 7% and 9% (P > 0.1), respectively, were low-level mupirocin resistant. No high-level mupirocin-resistant isolates were isolated from mupirocin (orthopaedic/vascular surgery) or elderly medicine control ward patients. There was no trend towards increasing prevalence of low-level mupirocin resistance during the four-year study period. The results of phage typing did not support the clonal spread of resistant strains. Long-term follow-up confirmed the efficacy of PPNMTT in reducing the prevalence of nasal carriage of S. aureus and MRSA in orthopaedic and vascular surgery patients. Despite four years of use of PPNMTT, there was no evidence of sustained emergence or spread of mupirocin resistance.     

Molecular surveillance of clinical methicillin-resistant Staphylococcus aureus isolates in neonatal intensive care units.

BACKGROUND AND OBJECTIVE: Methicillin-resistant Staphylococcus aureus (MRSA) has become an important nosocomial pathogen in our neonatal intensive care units (NICUs) and accounts for almost all S. aureus clinical isolates. The objective of this study was to assess the relatedness of these MRSA strains. DESIGN: MRSA clinical isolates were collected from infants hospitalized in our NICUs. Pulsed-field gel electrophoresis with SmaI digestion was used to fingerprint these isolates. SETTING: Level-III NICUs in a university-affiliated children's hospital. RESULTS: Between 1998 and 2000, a total of 122 MRSA clinical isolates were collected from 104 infants hospitalized in our NICUs. Fifteen infants had multiple isolates (range, 2 to 4 isolates). The sources of specimens included blood (72), pus (23), sputum (15), body fluids (3), and catheter tips (9). A total of 4 genotypes with 20 subtypes were identified. There were 2 genotypes in 1998, 2 genotypes in 1999, and 4 genotypes in 2000. All but 2 isolates belonged to either genotype A (63.1%; 7 subtypes) or genotype C (35.2%; 11 subtypes). Among the 15 infants with multiple isolates, the genotypes of the isolates from a single episode of MRSA infection were different in 2 of 12 cases, and reinfection with a new strain was noted in 3 of 5 cases with recurrent infections. CONCLUSIONS: Two predominant MRSA clones prevailed in our NICUs between 1998 and 2000. Polyclonal bacteremia and reinfection with a new strain were noted     

Methicillin-resistant Staphylococcus aureus, Hawaii, 2000-2002

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has generated considerable concern among medical and public health professionals. We used a statewide, population-based antimicrobial resistance surveillance system to examine epidemiologic trends for MRSA from outpatients and inpatients in Hawaii. Pediatric and adult patient populations were compared to assess characteristics of MRSA isolates specific for each group. From 2000 to 2002, 8,206 (26%) of 31,482 total S. aureus isolates were MRSA. During this period, the proportion of MRSA isolates increased in both outpatient and inpatient clinical settings (p<0.01). When stratified by age, annual trends showed a significant increase in the proportion of MRSA in adult patients (from 24% to 30%, p<0.01) but not in pediatric patients (from 25% to 27%, p>0.05). Although MRSA isolates from adults demonstrated high resistance to most non-beta-lactams, most MRSA isolates from pediatric outpatients remained susceptible to most non-beta-lactams.     

Discovery of torezolid as a novel 5-hydroxymethyl-oxazolidinone antibacterial agent

A series of novel substituted pyridyl phenyl oxazolidinone analogues were synthesized and their structure-activity relationship (SAR) was investigated based on in vitro and in vivo antibacterial activities. The minimum inhibitory concentrations (MICs) of the synthesized compounds against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) ranged from 0.12 to 2.0 μg/mL, and against Haemophilus influenzae (Hi) from 2.0 to 8.0 μg/mL. Compared to linezolid, only four compounds (11, 12, 21 and 29) showed higher in vitro antibacterial activities and better in vivo protective effects in mice. To improve the aqueous solubility, various prodrugs of compound 11 (DA-7157), which exerted a potency that was enhanced by 2-8-fold compared to that of linezolid, were synthesized. Among the prodrugs, the phosphate compound 42 exhibited excellent aqueous solubility (>50mg/mL in DW) and good pharmacokinetic profiles, along with better in vivo efficacy than linezolid. This compound 42 is currently undergoing clinical trials with the brand name Torezolid.     

Severe community-acquired pneumonia due to Staphylococcus aureus, 2003-04 influenza season

During the 2003-04 influenza season, 17 cases of Staphylococcus aureus community-acquired pneumonia (CAP) were reported from 9 states; 15 (88%) were associated with methicillin-resistant S. aureus (MRSA). The median age of patients was 21 years; 5 (29%) had underlying diseases, and 4 (24%) had risk factors for MRSA. Twelve (71%) had laboratory evidence of influenza virus infection. All but 1 patient, who died on arrival, were hospitalized. Death occurred in 5 (4 with MRSA). S. aureus isolates were available from 13 (76%) patients (11 MRSA). Toxin genes were detected in all isolates; 11 (85%) had only genes for Panton-Valentine leukocidin. All isolates had community-associated pulsed-field gel electrophoresis patterns; all MRSA isolates had the staphylococcal cassette chromosome mec type IVa. In communities with a high prevalence of MRSA, empiric therapy of severe CAP during periods of high influenza activity should include consideration for MRSA.     

Coagulase gemne variants associated with distinct populations of Staphylococcus aureus

An identifying characteristic of Staphylococcus aureus is the production of staphylocoagulase (coagulase). The aim of this study was to determine the clonal distribution of coagulase gene (coa) variants within populations of S. aureus defined by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and protein A variation. The N-terminal region of the coa gene from 43 methicillin-susceptible (MSSA) and 252 methicillin-resistant (MRSA) S. aureus human isolates and 9 animal S. aureus isolates was amplified and digested with HinfI. Twelve types were identified amongst the MSSA isolates and the majority (93%) of MRSA isolates were assigned to 5 of the 12 types. MLST and PFGE analysis identified epidemic populations of MRSA and each epidemic population was characterized by a different coagulase type. Nine of the 12 MLST-defined clonal complex ancestral genotypes recently described each carried a different coagulase type suggesting that coagulase evolution and the evolution of the clonal complexes are intimately related.     

Changing epidemiology of community-onset methicillin-resistant Staphylococcus aureus bacteremia.

OBJECTIVES: To review cases of community-onset Staphylococcus aureus bacteremia and to evaluate whether the risk factors and epidemiology of methicillin-resistant S. aureus (MRSA) bacteremia have changed from early reports. DESIGN: Retrospective case-comparison study of community-onset MRSA (n = 26) and methicillin-susceptible S. aureus (MSSA) (n = 26) bacteremias at our institution. SETTING: A 600-bed urban academic medical center. PATIENTS: Twenty-six patients with community-onset MRSA bacteremia were compared with 26 patients with community-onset MSSA bacteremia. Molecular analysis was performed on S. aureus isolates from the 26 MRSA cases as well as from 13 cases of community-onset S. aureus bacteremia from 1980 and 9 cases of nosocomial S. aureus bacteremia from 2001. RESULTS: The two groups were similar except that patients with MRSA bacteremia were more likely to have presented from a long-term-care facility (26.9% vs 4%; P = .05) and to have had multiple admissions within the preceding year (46% vs 15%; P = .03). Clamped homogeneous electric fields analysis of MRSA isolates from 1982 revealed predominantly that one clone was the epidemic strain, whereas there were 14 unique strains among current community-onset isolates. Among current nosocomial isolates, 3 patterns were identified, all of which were present in the community-onset cases. CONCLUSIONS: Previously described risk factors for MRSA acquisition may not be helpful in predicting disease due to the polyclonal spread of MRSA in the community. Unlike early outbreaks of MRSA in patients presenting from the community, current acquisition appears to be polyclonal and is usually related to contact with the healthcare system.