한국의 환자 및 건강인에서 분리한 E . coli 의 약제내성(藥劑耐性) 및 R Plasmids

설성용 대한미생물학회 1977 大韓微生物學會誌 Vol.12 No.1

家畜由來 大腸菌의 抗菌劑耐性 및 R plasmid

薛盛用,趙成萬,全燾基 대한화학요법학회 1984 대한화학요법학회지 Vol.2 No.2

韓牛, 乳牛, 豚 및 鷄等의 健康한 家畜 800마리의 糞使에서 分離한 總 2,034株의 大腸菌을 對象으로 15種 의 抗菌劑指에 對한 耐性檢査를 實施하여 耐性菌 및 R plasmid의 分布와 耐性樣相에 關하여 調査하였으며 아울러 非適合性에 의한 R plasmid의 分類, Col plasmid와 R plasmid와의 關係 및 非傳達性 R plasmid의 可動化에 關해서도 몇가지 實驗하여 다음과 같은 成績을 얻었다. 1. 健康한 家畜 800마리中 437마리(54.6%)에서 各種 藥劑에 耐性인 大腸菌을 保有하고 있었다. 家畜別家로는 鷄(94%)와 豚(85.5%)에서는 높은 頻度를 나타낸 反面, 韓牛(21%) 및 乳牛(18%)에서는 比較的 낮았다. 2. 傳達性脚 R plasmid 保有 大腸菌의 檢出率은 鷄 61.5%, 豚 55%였으나 乳牛(9.5%) 및 韓牛(2%)에서는 極히 낮았다. 3. 藥劑烈 耐性菌 出現率은 tetracycline(Tc)에 對한 耐性이 52.4%로 가장 높고 그 다음이 streptomycin(Sm), sulfisomidine(Su), ampicillin(Ap) 및 carbenicillin(Cb), chloramphenicol(Cm), kanamycin(Km)의 順이며 藥劑別 耐性菌 鉛境頻度는 家畜에 따라 현저한 差異를 나타내었다. Cephaloridine, cephalothin, gentamicin(Gm), 및 trimethoprim(쎄) 耐性菌은 드물었으며 Gm 및 Tp耐性인 菌은 豚 및 鷄에서만 分離되었다. Amikacin, colistin, nalidixic acid 및 rifampin에 耐性인 菌은 없었다. 4. Su, Ap, Cb, Km 및 Tp等에 耐性인 大部分의 菌株는 이들 藥劑에 對한 耐性을 最少發育沮止濃度가 512㎍/㎖f 以上으로 高度의 耐性을 나타내었다. Tc에 對한 家畜別 最少發育沮止濃度의 分布에는 有意한 差가 없었으나 Cm 및 Sm에는 현저한 差異를 보였으며 韓牛 및 乳牛由來菌보다 豚 및 鷄由菌讀예서 높았다. 5. 藥劑別 耐性傳達頻度는 Ap 템 Cb에서 65.6%로 가장 높고 그 다음으로 Cm(51.5%), Sm(47.5%), Km(45.9%), Tc(42.7%) 및 Su(37.2%)의 順으로 낮은 頻度를 보였다. 6. 分離菌의 耐性類型은 Tc, Sm, Su耐性型, Tc,Sm耐漢型 및 Tc?單劑耐性型이 大部分을 차지하고 있으며 豚과 鷄에서는 Tc, Sm, Su, Ap, Ct耐性型도 상당수 나타났다. 傳達後의 耐性類型은 家畜別 區分없이 Tc 單劑耐性型이 가장 많고 Tc, Sm耐性型, Sm, Su耐性型 및 Sm, Ap, Cb耐性型이 大部分을 차지하고 있었다. 7. 614株의 藥劑耐性 大腸菌中 78株(12.7%)가 colicin生産株였으며, 그 중 42株(53.8%)는 混合培養에 의해 耐性을 傳達시킬 수 있었다. 42株의 colicin生産株中 ll株에서는 colicin生産能과 藥劑耐性이 同時에傳達되었다. 8. 接合實驗에서 耐性이 薄達되지 않는 76株中 19株에서 ColB-K97 plasmid에 對해 混合培養에 의해 耐性의 一部 또는 全部가 最終被傳達菌에 傳達됨을 確認하였다. 9. 76個의 R plasmid에 對해 非適合性群에 의한 分類를 試圖하여 그중 30個의 R plasmid F Ⅰ, F Ⅱ, F Ⅳ 및 N非適合性群이 確認되었으나 鷄由來菌의 R plasmid는 모두가 N 非適合性群었다. Km, Gm 및 Tp에 耐性 R plasmid는 N와 F Ⅰ 非適合性群으로 分類되었다. 10. 非適合性에 의하여 한 菌株內에서 2個以上의 異種의 R plasmid의 共存與否를 確認한 바 豚由來의 한 菌株에서는 F Ⅰ 非適合性群과 分類不能의 R plasmid가 鷄由來 한 菌株에서는 N非適合性群과 分類不能의 R plasmid가 共存하고 있음이 確認되었으며 鷄由來 3株에서 分離된 서로 다른 耐性類型을 가지는 R plasmid는 모두 N 非適合性群으로 分類되어 deletion 結果로 나타나는 segregant로 생각되었다. A-t~tal of 2,034 sCrahs of E. coZi isolated from stools of 800 healthy domestic animals (Korean native cattle, dairy cattk, s~ine, and fowl) were tested for the resistance to 15 antimicrobial drugs aad distribution of. R plasmids.. In addition, classification of R plasmids by incompatibility groups, cc-transfer of Col plas&d with R plasmids, and mobilization of non-conjugative R plasaids were studied. 1. Of 800 healthy domestic animals, 431 (54.6%) excreted E. coZi resistant to one or more drugs. The prevalence of resistant strains was significantly higher in fod(94%] and swine (85.5%) than Korean native cattle (21%) and dairy 2. The frequencies of E. coli carrying conjugative R plasmids were 5541.5% in swine and fowl, whereas &hose were only 2-9.5% in Korean native and dairy cattle. 3. Of total isolaCes from healthy a n h d s , 52.4% were resistant to tetracyche(Tc). 43.4% to streptomycin(Sm), 38.9% to sulfisomidine(Su), 16.1 % to ampicillin(Ap) and carbenicillin (Cb), 10.4% to chloramphenicol(sJm), and 7.3% to kanamycinsm), wit6 considerable variations among animals in the frequency of resistance to drugs, Strains resistant to cephaloridme, cephalothin, gentamicin(Gm) and trhethoprirn(Tp3 were rarely encountered, and Gm-and Tp-resistant strains were isolated only from swine and fowL No strain was resistant to amikacii, colisth, nalidixic acid, and rifampin. 4. The majoritty of resistant strains had Mlcs of 512/Lg/ml or bigher for Su, Ap, Cb, Km, and Tp. There was a significant difference in the MIC distribution of Cm and Sm among isolates. MI& of drugs among isolates from swine and fowl were higher than isolates from Korean native and dairy caale, whereas no difference ma5 noted in MIC of Tc. 5. Resistance to A p and Cb was transferred most frequently(65.6%), and followed by Cm, Sm, Km,Tc, and Su in the decreasing order, ranging from 51.5% Eo 37.2%. Resistance to 'Tp and Gm was also mediated by conjugative R plasmids. 6. The most common resistance patterns were Tc Sm Su, Tc Sm, and Tc. The pattern of T c Sm Su Ap Cb was also observed frequently from swine and fowl. However, the most COE- man pattern transferred was Tc in all animal sources, and followed by Tc Sm, Sm Su, a i d Sm A? Cb. 7. Of 614 E. C O ~ Z : straixs resistant to hugs, 78(12.7%) produced coiicin, and conjugative R plasmids were detected in 42(53.8%> of these strains. In all of the colicin-producing strains .of E. coli, the transfer of Col plasmids were concurrent with the transfer of R plasmids. 8. In 19 strains of 76 which did not have auto-transmissible drug resistance, F-like plasmid CoiB-K98 Kas able to rcobilize ail 0:- part of the resistance markers. 9. The incornpatibiiity groups of '76 R plasmids were deternined with standard plasmids. Thirty R. plasmids were classified into incompatibility groups FⅠ , FⅡ , FⅣ, or N, and 6 R Tlasxi&s Fere not classified with the standard plasmids used. Incompatibility groups F Ⅰ . FⅡ. and F fii mere identified in strains from dairy cattle, incompatibility groups F 1 , F a . and N from swine, and incompatibiiity groilp N from fowl. X plasmids conferring resistance t o Rm, Gm, and Tp were classified into incompatibility group N and F 1. 10. The coexistence of two or more plasmids in single strain 7Tas tested, and it was fomd t h a t o3e strain from swine was infected with two plasmids: plasmid F I and untypable, ar,d one froin fowl contained plasmid N and untypable. On tbe other hand, seven plasmids obtained from three strains r i t h different resistance patterns n-ere indentified as incompatibility grou;, N, and some of then were considered as segregants of original plasmids from which some makers rrere deleted.

Salmonella 및 Shigella의 菌型 및 抗菌劑耐性의 推移

薛盛用 慶北大學校 醫科大學 1980 慶北醫大誌 Vol.21 No.1

1973年에서 1979年사이에 分離한 Salmonella와 Shigella의 菌型과 抗菌劑耐性의 推移를 보면 最近 S. paratyphi A의 感染이 增加하는 反面 S. typhi는 相對的으로 減少하는 傾向이 있었다. Shigella는 大部分이 Sh. flexneri였으며 漸次 增加하고 있던 Sh. sonnei가 1979年에는 전혀 分離되지 않는 反面 Sh. dysenteriae는 12株가 分離되었으며 그 중 10株가 type 1이었다. S. paratyphi A는 藥劑耐性菌이 거의 없으며 1973-1974年 相當數 分離되었던 藥劑耐性 S. typhi도 近年에는 分離되지 않았다. Shigella의 約 94%가 多藥劑耐性菌이며 最近 chloramphenicol, tetracycline, streptomycin, sulfonamide外에도 ampicillin 및 co-trimoxazole等에 多藥劑耐性인 菌이 增加하고 있으며 耐性菌의 約 78%가 R plasmid를 保有하고 있었다. (本硏究는 한곡장학금에 의하여 이루어졌음) Salmonella and Shigella isolated from patients with enteric infections during the period from 1973 through 1979 were serotyped and tested for the drug resistance. The majority of Salmonella isolated were S. typhi and the remaining was S. paratyphi A. There was a tendency of the gradual increase of S. paratyphi A in recent years. The majority of Shigella isolated were Sh. flexneri and approximately 17% was Sh. sonnei. Sh. sonnei increased gradually by years but no strain was isolated in 1979. Sh. dysenteriae was only rarely found. but 12 strains were isolated in 1979. Almost all strains of S. paratyphi A were susceptible to the drugs tested, while some S. typhi strains were multiply resistant to chloramphenicol (Cm), tetracydine (Tc), streptomycin (Sm), sulfonamides (Sa), ampicillin (Ap), and kanamycin, and the resistance was transferable to E. coli. Approximately 94% of shigellae isolated were multiply resistant to four or more drugs of Cm, To, Sm, Sa, Ap, trimethoprim (Tp), and nalidixic acid (Na); strains resistant to Tp were isolated in 1978 and 1979, and those resistant to Ap and Na increased sharply in recent two years. Nearly 78% of drug-resistant strains transferred their complete resistance except Na to E. coli.

1980年 大邱地方에서 分離한 Shigella의 菌型 및 抗菌劑耐性

薛盛用 慶北大學校 醫科大學 1980 慶北醫大誌 Vol.21 No.2

1980年 1月부터 12月 사이에 大邱地方에서 總 296株의 Shigella를 分離하였는바 菌型을 보면 總 296株中 206株(75%)가 Sh. flexneri였고 Sh. dysenteriae는 62株(21%) 였으며 Sh. sonnei는 27株(9%) 였고 Sh. bodyii는 type 4 1株 뿐이었다. Sh. dysenteriae는 46株가 type 1, 11株가 type 5, 5株가 type 2였고 Sh. flexneri는 184株가 2a였고 Y가 21株였다. 分離菌의 97%가 11種의 供試抗菌劑에 多藥劑耐性이 었으며 大部分이 5劑以上에 耐性이었다. 이들 菌은 主로 chloramphenicol, tetracycline, streptomycin 및 sulfidomidine의 4劑와 더불어 ampicillin, trimethoprim, nalidixic acid 등에 對한 耐性을 같이 가지고 있었고, kanamycin에 耐性인 것도 10株 있었다. Gentamicn, amikacin 및 cephalordine에 耐性인 것은 없었고 全供試藥劑에 感受性인것이 10株 있었다. A total of 296 strains of Shigella were isolated from stools of diarrheal patients of Taegu area during the period from January through December in 1980. Among them, 206 (75%) strains were classified as Shigella flexneri, 62 (21%) as Sh. dysenteriae, and 27 (9%) as Sh. sonnei. Only one strain was confirmed to be Sh. boydii 4. Sh. dysenteriae was classified into 46 strains of type 1 (Shiga type), 11 of type 5, and 5 of type 2. Sh. flexneri consisted of 184 strains of type 2a, 21 of variant Y, and one of type 4. Approximately 97% of strains isolated were multiply resistant to 11 antimicrobial drugs tested, and most of them were resistant to 5 or more drugs. Most strains were multiply resistant to four drugs of chloramphenicol, tetracycline, streptomycin, and sulfisomidine. In addition to these drugs, there were many strains resistant to one or more drugs of ampicillin, trimethoprim and/or nalidixic acid. There were 10 strains resistant to kanamycin in addition with other drugs. No strain was resistant to gentamicin, amikacin, and cephaloridine, and 10 strains were susceptible to all drugs tested.

blaTEM Hybridization을 이용한 Shigella sonnei에서 Ampicillin 내성의 분자역학적 분석

설성용,배홍경,우상열,정영숙,유학선,이유철,조동택 대한미생물학회 2004 Journal of Bacteriology and Virology Vol.34 No.1

R plasmid와 bla Gene을 이용한 병원 내 유행 ${\beta}$-lactam 내성 장내세균의 확인

설성용,원준희,김능희,유학선,이유철,조동택,김정완,Seol, Sung-Yong,Won, June-Hee,Kim, Neung-Hee,Yu, Hak-Sun,Lee, Yoo-Chul,Cho, Dong-Taek,Kim, Jung-Wan 대한미생물학회 2001 Journal of Bacteriology and Virology Vol.31 No.3

Clinical isolates of Enterobacteriaceae (189 Klebsiella, 61 Enterobacter, 32 Serratia, 19 E. coli, 7 Proteus, and 3 Citrobacter) from one university hospital were epidemiologically analyzed by using transferable R plasmids resistant ${\beta}$-lactam antibiotics including broad-spectrum cephalosporins. About 30% of E. cloacae and S. marcescens and about 5% of K. pneumoniae were resistant to one or more broad-spectrum ${\beta}$-lactam antibiotics including cefotaxim, ceftazidime, aztreonam, or cefoxitin but all isolates of E. aerogenes, K. oxytoca, and P. mirabilis were susceptible. Thirty-six conjugative R plasmids including 8 plasmids resistant expanded-spectrum cephalosporins were obtained from multiple resistant K. pneumoniae (19), E. cloacae (9), E. coli (4), and C. freundii (1). Thirty-one plasmids were subjected to R plasmid analysis and classified 20 different plasmid types. Among them 5, 2, and 2 plasmids belong to 3 different types respectively showed identical molecular size, endonuclease fragment pattern by Southern hybridization pattern by TEM-1 probe, pI value by isoelctric focusing, and also identical antibiogram and biotype of wild strains harboring plasmids. But all of plasmids resistant to cefotaxim, ceftazidime, aztreonam or cefoxitin showed different palsmid anlysis patterns. These results indicate that the epidemic strains of 3 clonal types had been present in this hospital and anlysis using transferable R plasmid and bla gene can be used to discriminate multi-resistant clinical isolates of Enterobacteriaceae.

Shigella의 비전달성 항균제 내성의 가동화

설성용,김상관,김정민,이제철,이상화,이유철,조동택 慶北大學校 醫科大學 1994 慶北醫大誌 Vol.35 No.1

목적 : 역학 및 계통 발생학적인 측면에서 chloramphenical(Cm), tetracycline(Tc), streptomycin (Sm), sulfisomidine(Su), ampicillin(Ap), trimethoprim(Tp) 내성형 을 나타내는 Shigella flexneri중 내성전달이 관찰되지 않는 균주를 대상으로 내성획득기전과 전달양상에 대하여 조사하였다. 재료 및 방법 : 균주 : Shigella flexneri중 항균제 감수성 검사에서 CmTcSmSuApTp에 중복내성을 나타내나, 내성전달이 관찰되지 않는 22주를 대상으로 Bochner법에 의한 내성제거와 transfer factor R64l를 이용하여 내성을 가동화시켜 원균주의 plasmid profile과 비교검토하였다. 결과 : 원균주와 내성제거균주를 대상으로 plasmid분리 및 전기영동하여 내성제거와 함께 plasmid band소실을 본 결과 13주에서 plasmid가 소실되었으나, 1주에서는 내성제거후에도 plasmid band소실이 관찰되지 않았다. 비전달성내성의 가동화실험에서 22주중 8주에서 14개의 plasmid가 가동화되었으며, CmTcSmSuApTp내성이며 97Mdal인 plasmid와 CmTcSmSuApTp내성이며 110Mdal인 plasmid는 FⅠ군으로, CmTcSmSuApTp내성이며 70Mdal인 plasmid는 FⅡ군으로 분류되었다. 결론 : 실험한 대부분의 균주에서 내성의 제거와 함께 plasmid의 소실이 관찰되었고, 가동화된 plasmid의 분자량도 원균주의 분자량과 거의 같았으며, 이중 일부분은 Shigella 전달성 R plasmid에서 흔히 발견되는 FⅡ나 FⅠ군으로 분류되는 것으로 보아 비전달성인 이들 R plasmid가 전달성 R plasmid 또는 transfer factor와 일시적 또는 영구적으로 결합하여 전달성 R plasmid로 발전되는 것으로 추측된다. Twenty-two Shigella flexneri strains resistant to chloramphenical(Cm), tetracycline(Tc), streptomycin (Sm), sulfisomidine(Su), ampicillin(Ap), and trimethoprim(Tp), but did not carry conjugative R plasmid, were tested for mobilization and elimination of resistance. Frequencies of elimination of drug resistance by Bochner's method were ranged from 5.3 to 100%. The plasmid profiles of mobilized and cured strains were compared with those of wild strains. Each of the wild strain showed one to three relatively large molecular weight plasmid( 146-59 Mdal) bands and was classified into 13 groups according to the plasmid profiles. On the other hand, thirteen cured strains lost one or two of these 128, 119, 100, 97, 94, and 58 Mdal plasmid indicating a nonconjugative resistance plasmid, but one strain showed the persistance of plasmid though the resistance was eliminated. From 8 strains among 22 wild strains, 14 plasmids were mobilized by transfer factor R641 and these mobilized plasmids were classified into Inc groups. Two 97 Mdal plasmids possessing CmTcSmSuApTp resistance and 110 Mdal plasmid possessing CmTcSmSuTp resistance were classified into Inc FⅠ group, and 70 Mdal plasmid possessing CmTcSmSuApTp resistance was Inc FⅡ group. But the remaining plasmids possessing CmTcSmSuApTp, SmSuApTp, SmSuTp, and Ap resistance could not be classified.

Shigella R plasmid의 비적합성에 의한 분류

설성용,이상화,김정완 대한화학요법학회 1988 대한화학요법학회지 Vol.6 No.1

1980년에서 1986년사이 분리된 838주의 Shigella를 대상으로 약제내성 양성의 변화와 내성전달, 그리고 비적합성에 의한 R plasmid의 분류를 시도하였다. Shigella flexneri의 가장 흔한 내성형은 chloramphenicol(Cm), tetracycline(Tc), streptomycin(Sm), sulfisomidine(Su), ampicillin(Ap), trime-thoprim(Tp)이고 Sh. sonnei에서는 CmTcSmSuTp형으로 ams 변화가 없었으나 1983년이래 Sh. flexneri에서 CmTcSmAp내성형이 되었다. 분리된 838주중 574주(69%)에서 1종 이상의 R plasmid로 보유하고 있었다. Shigella R plasmid의 대부분은 F Ⅱ 비적합성군으로 대개 그 내성양상은 분리균의 내성형과 동일하였다. 소수에서는 FⅠ,B 및 분류불능인 것이 관찰되었다. 표준 R plasmid로 분류불능인 25개의 R plasmid를 그들 상호간의 비적합성 실험에 의해 조사한 바 최소한 8가지 이상의 새로운 비적합성군이 존재하였으며 분자량은 120 megadalton에서 2.8 megadalton까지 다양하였다. A total of 838 strains of shigella isolated in years from 1980 to 1986 were studied for drug resistance pattern and R plasmid. The most prevalent resistant patterns of isolates and conjugative R plasmids were chloramphenicol(Cm) tetracycline(Tc) streptomycin(Sm) sulfisomidine(Su) ampicillin(Ap) trimethoprim(Tp) and CmTcSmSuTp in Sh. flexneri and sh. sonnei, respectively., the resistant pattern of CmTcSmAp in Sh. flexneri was frequently encountered since 1983 and in 1986, it was the predominant pattern. Approximately 69% of Shigella strains carried one or more conjugative R plasmids. The R plasmids from shigella were mostly beloned to Incompatibility group(INnc)FII and their resistance patterns were almost same as those of isolates, The R plasmids from small numbers of isolates were classified into Inc FI, Inc B, and untypable.

1982年 大邱地方에서 分離한 Shigella의 抗菌劑耐性 및 R plasmid

洩盛用,徐民濩,李裕澈 대한화학요법학회 1983 대한화학요법학회지 Vol.1 No.2

1982年 大邱地方에서 分離한 99株의 sigella의 菌型을 보면 69株(69.7%)가 S. flexneri였고, S. sonnei는 28株(28.3%) 였으며 2株가 Sh.dysenteriae였다. 分離된 99株中 79株가 2劑以上 藥劑에 耐性을 나타내었으며 大部分이 chloramphenicol (Cm), tetracycline (Tc), streptomycin (Sm), sulfisomidine (Su), ampicllin (AP) 및 trimethoprim (Tp) 等에 耐性을 나타내는 多劑耐性菌이었으나 S. sonnei는 모두가 Ap에 感受性이었다. 耐性菌의 大部分은 上記藥劑에 最少發育沮止濃度 256㎍/㎖ 또는 그以上의 高度耐性을 나타내었으며 Cm, AP 및 Cr等의 90% 發育沮止濃度는 S. flexneri와 S. sonnei사이에 현저한 差異를 보였는데 Cm은 Sh. sonnei가 Ap 및 Cr은 S. flexneri가 越等히 높았다. 少數(3~6株)는 kanamycin (Km), nalidixic acid (Na), rifampin (Rf) 및 Cr 等에 耐性이었으나 gentamicin 및 amikacin에 耐性인 菌'은 없었다. 耐性菌의 75.3%에서 Km, Na, Rf 및 Cr等을 除外한 모든 耐性이 單一類型으로 E, coil에 傳達되었으며 Km 耐性은 항상 따로 傳達되었다. The majority(69.776) of Shigella isolated in 1982 were Shigdla flexneri, the other were Sh. sonnei (28.3%) with only a small number of Sh. dysenterzae. Of 99 strains isoIated.97 were resistant to two or more drugs. Strains multiply resistant to s i x drugs of chloramphenicol (Cm) , tetracycline (Tc) , streptomycin (Sm), sulfisomidine (Su), ampicillin (Ap), and trimethoprim(Tp), were most frequently encountered but all Sh. sonnei were susceptible to Ap. MICs of the majority of resistant strains were more than 256,ug/ml to these drugs and there was a significant difference in MIC9o (needed to inhibit 90% of strains) of Cm, Ap, and ceph- aloridine (Cr) between Sh. fZexneri and Sh. sonnei; MICgo of Cm was higher in Sh. sonnei, wheras those of Ap and Cr were higher in Sh. flexneri. Strains resistant t o kanamycin (Km), nalidxic acid (Na), rifampin (Rf), and Cr were rarely encountered and no strain was resistant t o gentamicin and amikacin. The complete patterns of resistant to drugs except Km, Na, Rf, and Cr in approximately 75.3% of drug-resistant strains were co-transferred to Escherichia coli by conjugation and resistance t o Km was always transferred independently with other resistance.

bla<SUB>TEM</SUB> Hybridization을 이용한 Shigella sonnei에서 Ampicillin 내성의 분자역학적 분석

설성용(Sung-Yong Seol),배홍경(Hong-kyong Bae),우상열(Sang-Yel Woo),정영숙(Young-Sook Jeong),유학선(Hak-Sun Yu),이유철(Yoo-Chul Lee),조동택(Dong-Taek Cho) 대한미생물학회 2004 Journal of Bacteriology and Virology Vol.34 No.1