Journal of Korean Society of Oral Health Science 2016, January. 4(1): 10~16 ISSN: 2288-6079 부산지역치과병원의환경과종사자의원내감염세균오염 정소영, 박민아, 정혜인, 배일권 신라대학교치위생학과 Nosocomial Infectious Bacterial Contamination on Dental Hospital Environments and Staffs So Young Jung, Min-Ah Park, Hye-In Jung, Il Kwon Bae Dept. of Dental Hygiene, Silla University Correspondence to: Bae IK, Tel: +82-51-999-5430 Fax: +82-51-999-5707 E-mail: ikbae@silla.ac.kr Received December 15, 2015 Revised December 28, 2015 Accepted January 13, 2016 ABSTRACT Dental hospital environments and staffs can become contaminated with potentially variety pathogenic bacteria and have a possibility of causing cross infections. The aim of this study is to assess the identification and dissemination of dental hospital contaminated bacterial strains. We sampled computer keyboard, dental gown, light handle, mobile phone, nasal cavity, office phone, scaler, 3-way syringe, dental spittoon, handpiece, staff hands, and headrest. All the samples were cultured on blood agar plate (BAP) and MacConkey agar plate. The colonies grown on each plate were identified with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF) using MALDI Biotyper. Sixty-three strains on BAP growth that were identified as Arthrobacter castelli (n=1), Corynebacterium pseudodiphtheriticum (n=2), Corynebacterium propinquum (n=1), Lactobacillius fructivorans (n=1), Micrococcus luteus (n=2), Staphylococcus aureus (n=11), Staphylococcus capitis (n=2), Staphylococcus epidermidis (n=34), Staphylococcus haemolyticus (n=3), and Staphylococcus warneri (n=4). Sixty-nine strains on MacConkey agar plate were identified as Acidovorax delafieldii (n=3), Acidovorax temperans (n=3), A. baumannii (n=2), Acinetobacter johnsonii (n=3), Acinetobacter lwoffii (n=3), Acinetobacter pittii (n=3), Acinetobacter radioresistens (n=1), Acinetobacter spp. (n=1), Chryseobacterium spp. (n=1), Citrobacter freundii (n=2), Comamonas testosteroni (n=1), Cupriavidus metallidurans (n=1), Delftia acidovorans (n=1), Enterobacter aerogenes (n=6), Enterobacter cancerogenus (n=1), Klebsiella oxytoca (n=2), K. pneumoniae (n=4), Klebsiella spp. (n=1), Moraxella osloensis (n=2), Moraxella spp. (n=2), P. aeruginosa (n=4), Pseudomonas asplenii (n=1), Pseudomonas entomophila (n=1), Pseudomonas jessenii (n=1) Pseudomonas luteola (n=1), Pseudomonas monteilii (n=2), Pseudomonas oryzihabitans (n=1), Pseudomonas rhodesiae (n=3), Rhizobium tumefaciens (n=1), Serratia marcescens (n=2), Sphingomonas paucimobilis (n=1), Sphingomonas spp. (n=3), Stenotrophomonas maltophilia (n=2), and Stenotrophomonas spp. (n=2). Our results showed that dental hospital environments and staffs were contaminated with variety nosocomial bacteria. Key words: Dental hospital, Cross infection, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry, Contamination, Bacteria Copyright@ 2016 by the Korean Society of Oral Health Science (KOHS) 10
Nosocomial Infectious Bacterial Contamination on Dental Hospital Environments and Staffs I. 서론 우리가생활하는대부분의장소는세균이존재하며, 이러한현상은병원환경을비롯한치과병원이라고해서예외가될수는없다. 환기가적절하게이루이지지않은밀폐된치과병원의환경에서서식하는세균들은적절한온도와습도가유지될경우증식하여의료진의손, 가운, 등을통하여확산될수있다. 또한치과병원에서이루어지는각종의료서비스를통하여감염세균들이환자에서의료진으로, 의료진에서환자로, 환자에서환자로및의료진에서의료진으로확산되는등병원내감염 ( 교차감염 ) 이발생할수있다. 최근미국의질병관리통제센터 (CDC, Center for Disease Control and Prevention) 에서는이러한병원감염에의해서만미국에서연간약 170만건의원내감염이발생하고이가운데약 10만명이사망한다고하였으며이를통해약 40억달러의의료비용이소모된다고보고하였다 [1]. 주요원내감염증의원인이되는세균은 methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus spp. (VRE), carbapenem-resistant Acinetobacter baumannii (CRAB), carbaepenem-resistant Pseudomonas aeruginosa (CRPA), carbaepenem-resistant Enterobacteriaceae (CRE), 장관내독소생성 Clostridium difficile 등이있다. 이러한세균가운데의료관련감염병다제내성균 6종 (MRSA, VRE, MRAB, MRPA, CRE 및 vancomycin-resistant S. aureus) 에대해서우리나라의질병관리본부에서도법정감염병중지정감염병으로지정하여관리하고있다 [2]. S. aureus는원내감염의가장흔한원인균으로면역력이저하된입원환자의상처부위및연조직감염증을통해급성폐렴이나패혈증으로악화될뿐아니라 MRSA의경우감염시항균제의선택압상승으로치료가지연되어유병률과사망률이증가한다 [3-6]. MRSA는확산속도가매우빨라첫번째보고가있은후, 10년만에전세계에확산되었고국내에도출현하였다 [7]. 이에국내에서는 Korean Nationwide Surveillance of Antimicrobial Resistance (KONSAR) 그룹을통해서 MRSA에대한표본감시를진행중으로현재, 국내에서이세균의분리율을약 69% 로매우높다 [8]. VRE는 Enterococci가주로 vana 유전자를획득한것으로미국의 CDC에서는입원환자의변또는비강에대한 active surveillance의수행을권장할정도로감염관리에매우중요한세균이다 [9][10]. CRAB는주로 class D carbapenemase (OXA-23에서 27, OXA-40 등 ) 를생성하여 imipenem, meropenem 등의항균제에내성을획득한것으로국내에서분리되는 CRAB 대부분은 OXA-23을생성에의한것이며동일한 clonal complex (CC) 로알려져있다 [11][12]. 이세균의대한연구가시행될 1998년에국내의 CRAB 분리율은 5% 였으나최근의조사에서는약 70% 로과거에비해현저하게증가하였다 [12][13]. MRPA는 class B metallo-β-lactamase를생성하는균주와세균의염색체에존재하는 class C β -lactamase의과발현, 세포외막에존재하는 porin의소실및변화그리고 efflux pump의복합작용으로내성을획득한균주로나눠지며국내에서분리되는세균의약 60% 가이세균인것으로보고되고있다 [14][15]. CRE의국내보고는 2003년에분리된 GES-5 생성 Klebsiella pneumoniae를필두로최근에는 KPC-2, MBL 생성등이보고되고있으며이세균의확산이감염관리의주요한관심이되고있다 [16][17][18]. 하지만이러한세균들가운데 MRSA를제외하고국내의치과병원에서분리된세균을보고한사례는매우드물며분리된세균들에대한전체적인균종동정의보고도흔하지않다 [19]. 본연구에서는부산지역의치과병원의환경과종사자에게서분리된세균의균종을동정하여이들세균의균종분포를확인하여향후, 치과병원에서교차감염및내성획득가능성을확인하고자한다. II. 재료및방법 1. 샘플의채취및보관 본연구를위해부산지역 10개의치과병 의원에근무하는종사자및환경에서샘플을채취하였다. 채취부위는종사자의경우비강, 손, 휴대전화, 가운등이었고환경은라이트손잡이, 3-way syringe, headrest, handpiece, scaler, 타구대, 병원전화기, 컴퓨터키보드등이었다. 샘플의채취는먼저 sodium chloride, postssium chloride, calcium chloride, magnesium chloride, disodium phosphate, sodium thioglycollate, monopotassium phosphate, agarose 및멸균증류수가포함된 ph7.3±0.2 ( 상온 ) 의 amies 수송배지 (Micromedia, Busan, Korea) 를이용하였다. 수송배지의면봉을멸균증류수에적시고샘플을채취할부분에문지르거나긁은 11
Journal of Korean Society of Oral Health Science Vol. 4 No. 1 January 2016 후, 면봉의머리부분이 amies 수송배지에깊이박히도록찔러보관하였으며, 채취된샘플은가급적빨리실험실로옮겨증균배지에배양하였다. 사용된증균배지는혈액한천배지와 MacConkey 한천배지를이용하였고증균된집락의개수, 크기, 모양, 색등을바탕으로우선적인균종을분리하여초저온냉동고에저장하였다. 2. 분리된세균의균종확인평판배지증균양상을바탕으로수집된세균은평판배지에순수배양하여이온질량분석법의하나인 matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS, Bruker Daltonic GmbH, Leipzig, Germany) 를통하여균종을동정하였다. III. 결과 본연구를통하여비강 58개, 휴대전화 55개, 치과환경 99 개등의샘플을수집하였으며그람양성세균 63주와그람음성세균 71주가동정되었다. 동정된그람양성세균은 Arthrobacter castelli 1주, Corynebacterium pseudodiphtheriticum 2주, Corynebacterium propinquum 1주, Lactobacillius fructivorans 1 주, Micrococcus luteus 2주, S. aureus 11주, Staphylococcus capitis 2주, Staphylococcus epidermidis 34주, Staphylococcus haemolyticus 3주, Staphylococcus warneri 4주이었고그람음성세균은 Acidovorax delafieldii 3주, Acidovorax temperans 3주, A. baumannii 2주, Acinetobacter johnsonii 3주, Acinetobacter lwoffii 3주, Acinetobacter pittii 3주, Acinetobacter radioresistens 1주, Acinetobacter spp. 1주, Chryseobacterium spp. 1주, Citrobacter freundii 2주, Comamonas testosteroni 1주, Cupriavidus <Table 1> Numbers of dental hospital environments and staffs contaminated with nosocomial infectious bacteria Source (n) Gram positive strains (n) Gram negative strains (n) Computer keyboard (6) Micrococcus luteus (1), Staphylococcus warneri (1), Staphylococcus haemolyticus (1) Citrobacter freundii (2), Pseudomonas aeruginosa (1) Dental gown (9) Micrococcus luteus (1), Staphylococcus capitis (1) Acinetobacter pittii (1), Acinetobacter lwoffii (1), Acinetobacter radioresistens (1) Pseuomonas asplenii (1), Pseudomonas oryzihabitans (1), P. aeruginosa (1), Pseudomonas monteilii (1) Light handle (4) Staphylococcus epidermidis (1), Lactobacillus fructivorans (1) Moraxella spp. (1), A. lwoffii (1) Mobile phone (16) Nasal cavity (70) Office phone (6) S. warneri (1) Corynebacterium propinguum (1), S. capitis (1), S. epidermidis (4) Scaler (4) Arthrobacter castelli (1) Corynebacterium pseudodiphtheriticum (2), M. luteus (2), Staphylococcus aureus (11), S. epidermidis (29), S. haemolyticus (2), S. warneri (2) Acinetobacter johnsonii (2), Moraxella osloensis (1), A. lwoffii (2), A. pittii (1), Comamonas tertosteroni (1), Pseudomonas rhodesiae (1), Pseudomonas jessenii (1), Serratia marcescens (1) Acinetobacter baumannii (2), Enterobacter aerogenes (6), Klebsiella spp. (1), Klebsiella oxytoca (2), Klebsiella pneumoniae (4), M. osloensis (1), P. monteilii (1), P. rhodesiae (2), S. marcescens (1), Sphingomonas spp. (1), Stenotrophomonas maltophilia (1) Moraxella spp. (1), Pseudomonas luteola (1), Sphingomonas spp. (2) Acidovorax temperans (1), Acidovorax delafieldii (1), Cupriavidus metallidurans (1) 3-way syringe (3) A. temperans (2), Delftia acidovorans (1) A. pittii (1), A. johnsonii (1), Rhizobium tumefaciens (1), Dental spittoon (8) P. aeruginosa (1), Pseudomonas entomophila (1), Stenotrophomonas maltophila (1), Sphingomonas spp. (2) A. temperans (2), Chryseobacterium spp. (1), Sphingomonas Handpiece (5) paucimobilis (1), P. aeruginosa (1) Hands (2) Acinetobacter spp. (1), Enterobacter cancerogenus (1) 12
Nosocomial Infectious Bacterial Contamination on Dental Hospital Environments and Staffs metallidurans 1주, Delftia acidovorans 1주, Enterobacter aerogenes 6주, Enterobacter cancerogenus 1주, Klebsiella oxytoca 2주, K. pneumoniae 4주, Klebsiella spp. 1주, Moraxella osloensis 2주, Moraxella spp. 2주, P. aeruginosa 4주, Pseudomonas asplenii 1주, Pseudomonas entomophila 1주, Pseudomonas jessenii 1주 Pseudomonas luteola 1주, Pseudomonas monteilii 2주, Pseudomonas oryzihabitans 1주, Pseudomonas rhodesiae 3주, Rhizobium tumefaciens 1주, Serratia marcescens 2주, Sphingomonas paucimobilis 1주, Sphingomonas spp. 3주, Stenotrophomonas maltophilia 2주, Stenotrophomonas spp. 2주가확인되었으며달리분류되지않는장내세균 2주와 MALDI-TOF MS에의해서동정되지않은균주가 28주이었다. 분리된세균의채취한위치는종사자의비강 (n=70) 에서그람양성 48주, 그람음성 22주로가장많이분리되었고다음으로종사자의휴대전화 (n=16), 가운 (n=9), 타구대주변 (n=8), 키보드 (n=6), 전화기 (n=6), handpiece (n=5), 라이트손잡이 (n=4), scaler (n=4), 3-way syringe (n=3), 종사자의손 (n=2) 등의순서로분리되었으며 headrest에서분리된한균주는동정에실패하였다 <Table 1>. IV. 고찰및결론 지금까지미생물을동정하는방법으로전통적인생화학적방법과유전자의염기서열분석이흔히활용되어져왔으나최근에는 MALDI-TOF MS를이용한이온질량분석법이적용되어지고있다. MALDI-TOF MS는 matrix와혼합된시료를레이저섬광으로이온화시키고이때발생한이온들이진공관을통과하여탐지기에도달하는시간을측정하여질량을분석하는것으로일반적인세균의동정뿐아니라결핵균, 진균등의동정에도널리활용되고있다 [20][21]. 분리된세균은그람양성 5속 10종, 그람음성 15속 34종으로전체 134주가동정되었다. 이가운데그람양성균의 S. aureus 를비롯한 Staphylococcus spp. 가 54주로가장흔하였다. S. aureus 균종은병원감염의가장흔한세균으로일반인의비강에존재하는정상상재균이지만 methicillin에내성을획득한이세균이 Mycoplasma pneumoniae에의한폐렴환자에서인후종양의원인이되었고 [3][22], 일반적인충치부위 (gingivitis, periodontitis) 에서도발견되는등 [23] 구강환경의건강여부와 무관하게분리되기도하였다 [24]. Corynebacterium spp. 은자연계에널리존재하는세균으로디프테리아를유발하는 Corynebacterium diphtheriae가알려져있으나본연구에서는 C. pseudodiphtheriticum (2주) 과 C. propinquum (1주) 가분리되었다 [25]. 이두균종은구강내상재세균으로일본에서호흡기감염증을일으킨사례가보고되었을뿐구강감염사례는드물다 [26]. Micrococcus spp. 와 Arthrobacter spp. 는토양이나먼지에서주로분리되는세균으로최근임상검체에서분리된사례가있을뿐이들에대한연구는부족한실정이다 [27][28]. Lactobacillius fructivorans는당을분해하여유산을생성하는유산균으로인간의구강을비롯한장관과여성의질에존재하여외부에서유입되는세균에의한감염증을예방하는활생균 (probiotics) 으로알려져있다 [29]. 동정된그람음성세균 69주가운데 51주 (73.9%) 가 glucose non-fermenting gram-negative bacillus (GNFB) 로확인되었다. 균종별로 Pseudomonas spp. 와 Acinetobacter spp. 가각각 14주, Acdovorax spp. 6주, Moraxella spp., Sphingomonas spp., Stenotrophomonas spp. 각 4주였으며 Chryseobacterium spp., Comamonas spp., Cupriavidus spp., Delftia spp. 및 Rhizobium spp. 가각 1주씩으로확인되었다. 이들 GNFB는토양과물등자연환경과병원환경그리고인체의피부에서도상재하는세균으로기회감염균이기도하다 [15][30]. 대표적으로 Pseudomonas spp. 는 penicillin을비롯한다양한 β-lactam 항균제에자연내성이고다양한항균제에내성을쉽게획득하여임상에서큰위협이되는세균으로알려져있으며 P. aeruginosa가가장흔하다 [31][32]. 또한 carbapenem을비롯한다양한광범위항균제에내성을획득한이세균의국내확산은병원감염관리에큰위협이되고있다 [15]. Acinetobacter spp. 는국내에서는 OXA-23을생성하는 CC 92 균주의확산으로 carbapenem 내성율이급격히증가하였고이를계기로질병관리본부의지정감염병이되었으며전세계적으로이세균에의한감염증발병사례가크게증가하고있다 [12][33]. Stenotrophomonas spp. 는일반적인감염증치료제로사용하는다양한항균제에내성을보이는것으로보고되고있다 [34]. 아마이세균은의료장비에집락화되어있다가 catheter관련감염, 균혈증, 요로감염등을일으키는것으로생각된다 [34]. 그람음성세균의 GNFB를제외한 18주는모두 Enterobacteriaceae로확인되었으며이들은 Klebsiella spp. 와 Enterobacter spp. 가각 7주, Citrobacter spp. 와 Serratia spp. 가 13
Journal of Korean Society of Oral Health Science Vol. 4 No. 1 January 2016 각 2주였다. 분리된 Klebsiella spp. 가운데 4균주는 K. pneumoniae ( 폐렴간균 ) 으로이세균은과거에알콜중독자나당뇨환자에게지역사회획득성폐렴의원인균으로알려졌으며, 최근에는 KPC carbapenemase를생성하는장내세균의대표적인병원감염증원인균으로질병관리본부의법정감염병중지정감염병에포함되어있다 [2][35][36]. Enterobacter spp. 가운데 E. aerogenes가 6주로가장많이분리되었다. Enterobacter spp. 는중요한병원감염의원인균으로국내에서 chromosomal AmpC β-lactamase 생성과동시에 outer membrane protein (OmpF) 의결실로인한 carbapenem 항균제내성등이보고되었으나이세균에의한구강감염증사례는극히드물다 [18]. Serratia marcescens 2균주는한종사자의비강과휴대전화에서동시에분리되어같은세균임을의심되어졌다. 이세균도 Enterobacter spp. 의사례와마찬가지로 chromosomal AmpC β -lactamase의과발현과동시에 OmpF의소실에의해 carbapenem 계항균제인 meropenem을회득한사례가있으며프랑스의혀복원 (tongue reconstruction) 환자에서 extended-spectrum β -lactamase SHV-12를생성하는이세균에의한감염사례가보고된바있다 [37]. 본연구를통하여부산지역치과병원종사자와병원환경에존재하는세균의균종과분리되는세균의빈도를확인할수있었다. 분리된세균중에는병원감염의중요한원인균으로일반병원에서도감염관리에위협이되고있는 S. aureus, Pseudomonas spp., Acinetobacter spp. 및 Enterobacteriaceae가포함되었다. 최근에는이러한세균들이병원내에서항균제와소독제에내성을획득하여임상에서이루어지는의료행위의성공에큰방해요인으로작용하고있어적극적인감염관리가요구된다. 하지만종합병원을비롯한대형병원과는달리치과병원은대부분비교적소규모이고정부의감염관리모니터링에도배제되어있는사각지대에놓여있는것이현실이며적극적인감염관리는물론이거니와소극적인감염관리시스템의도입에도풀어야할난제가산적해있다. 이러한어려움을극복하여치과종사자와환자의안전을지키기위해서다양한학문적인접근을통해문제를과학적으로해결해야만할것으로판단된다. 참고문헌 1. http://www.cdc.gov/ncidod/dhqp/hai.html 2. http://www.cdc.go.kr/cdc/main.jsp 3. Boucher HW, Corey GR: Epidemiology of methicillin-resistant Staphylococcus aureus. Clin Infect Dis 46(Suppl 5): S344 9, 2008. 4. Calfee DP: Methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, and other Gram-positives in healthcare. Curr Opin Infect Dis 25(4): 385-94, 2012. 5. Köck R, Becker K, Cookson B, et al: Methicillin-resistant Staphylococcus aureus (MRSA): burden of disease and control challenges in Europe. Euro Surveill 15(41): 19688, 2010. 6. Moellering RC Jr: MRSA: The first half century. J Antimicrob Chemother 67(1): 4-11, 2012. 7. Lee K, Chang CL, Lee NY, Kim HS, Hong KS, Cho HC. Korean Nationwide Surveillance of Antimicrobial Resistance of Bacteria in 1998. Yonsei Med J 41(4): 497 506, 2000. 8. Lee K, Kim MN, Kim JS, et al: Further increases in carbapenem-, amikacin-, and fluoroquinolone-resistant isolates of Acinetobacter spp. and P. aeruginosa in Korea: KONSAR study 2009. Yonsei Med J 52(5): 793 802, 2011. 9. Courvalin, P: Resistance of Enterococci to glycopeptides. Antimicrob Agents Chemother 34(12): 2291-96, 1990. 10. Sigeg JD, Rhinehart E, Jackson M, Chiarello L: Management of multidrug-resistant organisms in healthcare settings, 2006. Bethesda, MD: Centers for Diesease Control and Prevention; 2006. 11. Yigit H, Queenan AM, Anderson GJ, et al: Novel carbapenem-hydrolyzing β-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob Agents Chemother 45(4):1151-61, 2001. 12. Lee Y, Lee J, Jeong SH, Lee J, Bae IK, Lee K: Carbapenem-non-susceptible Acinetobacter baumannii of sequence type 92 or its single-locus variants with a G428T substitution in zone 2 of the rpob gene. J Antimicrob Chemother 66(1): 66-72, 2011. 13. Chong Y, Lee K: Present situation of antimicrobial 14
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Journal of Korean Society of Oral Health Science Vol. 4 No. 1 January 2016 caused by gram-negative bacilli. Clin Infect Dis 41(6): 848-54, 2005. 34. Looney WJ, Narita M, Mühlemann K: Stenotrophomonas maltophilia: an emerging opportunist human pathogen. Lancet Infec Dis 9(5): 312-23, 2009. 35. Vuotto C, Longo F, Balice MP, Donelli G, Varaldo PE: Antibiotic resistance related to biofilm formation in Klebsiella pneumoniae. Pathogens 3(3): 743-58, 2014. 36. Gupta N, Limbago BM, Patel JB, Kallen AJ: Carbapenem-resistant Enterobacteriaceae: epidemiology and prevention. Clin Infect Dis 2011; 53: 60-67. 37. Dhawan B, Bonnet R, Shukla NK, Mathur P, Das BK, Kapil A: Infection with an extended-spectrum β -lactamase-producing of Serratia marcescens following tongue reconstruction. J Clin Microbiol 41(5): 2233-4, 2003. 16