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  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/298054475 Characterization of clinical Staphylococcus aureus isolates harboring mecA orPanton-Valentine leukocidin genes from four tertiary care hospitals inIndonesia  Article   in  Tropical Medicine & International Health · March 2016 DOI: 10.1111/tmi.12692 CITATIONS 6 READS 182 15 authors , including: Some of the authors of this publication are also working on these related projects: Respiratory infections   View projectFraksinasi amilase dari Endomycopsis fibuligera ITBRcc64 dengan metode presipitasi amonium sulfat   View projectDewi SantosaningsihBrawijaya University/dr.Saiful Anwar hospital, Malang, Indonesia 12   PUBLICATIONS   33   CITATIONS   SEE PROFILE Nyoman S BudayantiUdayana University 17   PUBLICATIONS   80   CITATIONS   SEE PROFILE Kuntaman KuntamanAirlangga University 35   PUBLICATIONS   458   CITATIONS   SEE PROFILE Alex van BelkumBioMérieux 785   PUBLICATIONS   27,500   CITATIONS   SEE PROFILE All content following this page was uploaded by Nyoman S Budayanti on 07 May 2016. The user has requested enhancement of the downloaded file.  Characterisation of clinical  Staphylococcus aureus  isolatesharbouring  mecA  or Panton  –  Valentine leukocidin genes fromfour tertiary care hospitals in Indonesia Dewi Santosaningsih 1 , Sanarto Santoso 1 , Nyoman S. Budayanti 2 , Ketut Suata 2 , Endang S. Lestari 3 , HendroWahjono 3 , Aziz Djamal 4 , Kuntaman Kuntaman 5 , Alex van Belkum 6,7 , Mitchell Laurens 6,8 , Susan V. Snijders 6 ,Diana Willemse-Erix 6,9 , Wil H. Goessens 6 , Henri A. Verbrugh 6 and Juli € ette A. Severin 6 1  Department of Microbiology, Faculty of Medicine, Brawijaya University/Dr.Saiful Anwar Hospital, Malang, Indonesia 2  Department of Microbiology, Faculty of Medicine, Udayana University/Sanglah Hospital, Denpasar, Bali, Indonesia 3  Department of Microbiology, Faculty of Medicine, Diponegoro University/Dr.Kariadi Hospital, Semarang, Indonesia 4  Department of Microbiology, Faculty of Medicine, Andalas University/Dr.M.Djamil Hospital, Padang, Indonesia 5  Department of Microbiology, Faculty of Medicine, Airlangga University/Dr.Soetomo Hospital, Surabaya, Indonesia 6  Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Centre, Rotterdam, the Netherlands 7  Microbiology Unit, Biom   erieux, Inc., La Balme, France 8  BaseClear BV, Leiden, the Netherlands 9  Molecular Diagnostics, Jeroen Bosch Hospital, Tilburg, the Netherlands Abstract  objectives  To determine the prevalence, antimicrobial susceptibility profiles and clonal distributionof either methicillin-resistant  Staphylococcus aureus  (MRSA) or Panton  –  Valentine leukocidin (PVL)-positive  S. aureus  obtained from clinical cultures in Indonesian hospitals. methods  S. aureus  isolates from clinical cultures of patients in four tertiary care hospitals inDenpasar, Malang, Padang and Semarang were included. We assessed the antimicrobial susceptibilityprofiles using the Vitek2  system, determined the presence of the  mecA  gene and genes encoding PVLusing PCR and analysed the clonal relatedness with Raman spectroscopy. SCC mec  typing wasperformed for all MRSA isolates. Multilocus sequence typing (MLST) was performed for a subset of isolates. results  In total, 259  S. aureus  strains were collected. Of these, 17/259 (6.6%) and 48/259 (18.5%)were MRSA and PVL-positive methicillin-susceptible  S. aureus  (MSSA), respectively. The prevalence of MRSA and PVL-positive MSSA ranged between 2.5  –  8.9% and 9.5  –  29.1%, respectively and dependedon geographic srcin. PVL-positive MRSA were not detected. Raman spectroscopy of the strainsrevealed multiple Raman types with two predominant clusters. We also showed possible transmissionof a ST239-MRSA-SCC mec  type III strain and a ST121 PVL-positive MSSA in one of the hospitals. conclusions  We showed that MRSA and PVL-positive MSSA are of clinical importance inIndonesian hospitals. A national surveillance system should be set-up to further monitor this. Toreduce the prevalence of MRSA in Indonesian hospitals, a bundle of intervention measures is highlyrecommended. keywords  Asia, Indonesia, methicillin-resistant  S. aureus , panton  –  valentine leukocidin, Staphylococcus aureus Introduction Staphylococcus aureus  is recognised as an importantpathogen, both in the hospital and community settings[1]. The emergence and spread of methicillin-resistant S. aureus  (MRSA), covering both hospital-associatedMRSA (HA-MRSA) and community-associated MRSA(CA-MRSA), has been a major problem worldwide [2  –  6].Traditionally, HA-MRSA has been associated with mul-tidrug resistance and staphylococcal cassette chromosome mec  (SCC mec ) types I, II and III, while CA-MRSA hasbeen associated with SCC mec  types IV and V and thepresence of Panton  –  Valentine leukocidin (PVL) genes.However, this distinction has blurred in many countriesas the CA-MRSA clones have been introduced into thehospital and the HA-MRSA in the community [7]. 610  © 2016 John Wiley & Sons Ltd Tropical Medicine and International Health doi:10.1111/tmi.12692 volume 21 no 5 pp 610  –  618 may 2016  S. aureus  infections, including MRSA infections, havelong been underappreciated in resource-limited countriesin south and east Asia, where the healthcare agenda pri-oritises other healthcare issues [8]. With the rapid emer-gence of antimicrobial resistance worldwide, this topichas, however, increasingly gained interest. Still, only lim-ited data are available on the epidemiology of   S. aureus infections in Indonesia. We have recently shown thatMRSA was carried among patients screened at dischargefrom hospital in Indonesia, but with significant geograph-ical variation [9]. Among the MRSA strains, the HA-MRSA clone sequence type (ST) 239-SCC mec  III pre-vailed. In addition, a high prevalence of PVL was foundamong carriage and community-onset infectious strainsof methicillin-susceptible  S. aureus  (MSSA) from Indone-sia [9  –  13]. However, data on clinical  S. aureus  fromIndonesian hospitals are lacking. In this study, we deter-mined the prevalence, antimicrobial susceptibility profilesand clonal distribution of either MRSA or PVL-positive S. aureus  obtained from clinical cultures in four Indone-sian hospitals. Materials and methods Setting Four tertiary care hospitals located on Sumatra island, Java island and Bali island (Figure 1) participated inthis study: Sanglah Hospital in Denpasar (Bali; 704beds), Dr. Saiful Anwar Hospital in Malang (East Java;810 beds), Dr. M. Djamil Hospital in Padang (WestSumatra; 688 beds) and Dr. Kariadi Hospital in Semar-ang (Central Java; 779 beds). The study was performedfrom January 2008 to January 2009 in Padang andDenpasar, from October 2009 to January 2010 inMalang and from April 2008 to October 2009 inSemarang. This study was approved by the medicalethics committee of Dr. Saiful Anwar Hospital(Malang), Sanglah Hospital (Denpasar) and Dr. KariadiHospital (Semarang) related to the ‘MRSA study’ inIndonesia. Isolates were obtained as part of routinediagnostic testing and analysed anonymously for thisstudy. Bacterial isolates S. aureus  strains were isolated and identified by clinicallyindicated culture in each hospital involved in this study. S. aureus  from all departments were included. Isolateswere stored in trypticase soy agar until further characteri-sation could be performed. Confirmation of identificationand antibiotic susceptibility testing of the  S. aureus strains were carried out by the Vitek2  system(bioM  erieux, Marcy l’Etoile, France). Antibiotics testedincluded macrolides (clindamycin, erythromycin), amino-glycosides (gentamicin, tobramycin), fluoroquinolones(ciprofloxacin, levofloxacin and moxifloxacin), glycopep-tides (teicoplanin, vancomycin), trimethoprim  –  sul-famethoxazole, fosfomycin, fusidic acid, linezolid,mupirocin, nitrofurantoin, rifampicin and tetracycline.Only one clinical culture with  S. aureus  per patient wasincluded in this study. DNA isolation and detection of   mecA  and PVL genes Bacterial DNA was isolated using the MagNa Pure LCDNA system (DNA isolation kit III; Roche MolecularBiochemicals, Mannheim, Germany) [14]. The DNA PadangJakartaSemarangMalangDenpasar Figure 1  Map of Indonesia depicting the four cities involved in this study (squares). The capital city Jakarta is also indicated (circle). © 2016 John Wiley & Sons Ltd  611 Tropical Medicine and International Health  volume 21 no 5 pp 610  –  618 may 2016 D. Santosaningsih  et al.  Staphylococcus aureus  in Indonesia  concentration was measured spectrophotometrically, andthe samples were stored at   20  ° C. PCRs to detect  mecA and PVL ( lukF-PV and lukS-PV  ) genes were performedas previously described [15, 16]. SCC mec   typing Multiplex PCR to characterise the SCC mec  of   S. aureus harbouring the  mecA  gene was conducted as previouslydescribed [17]. Raman spectroscopy The clonal relationship among MRSA and PVL-positiveMSSA isolates was analysed using Raman spectroscopy(SpectraCell RA  Bacterial Strain Analyzer, RiverD interna-tional BV, Rotterdam, The Netherlands), as previouslydescribed [18]; [19]; [9, 20].Raman spectral analysis was performed using Spec-traCell RA  software version 1.9.0.13444:24 (RiverD interna-tional). The squared Pearson correlation coefficient (R 2 )determined the similarity of the sample spectra and theknown R 2 distribution of the identical and unrelated strains.In five different measurements, we included the ATCC(American Type Collection Culture) 43300 strain as a repro-ducibility control. A two-dimensional plot was created tocompare the similarity of multiple isolates; the similarity of two isolates was presented by a colour scale. The clonal relat-edness was determined by setting the similarity threshold andcut-off value as previously described [9]. Multilocus sequence typing (MLST) Fourteen  S. aureus  isolates were further analysed byMLST to allow international comparison [21]. From eachRaman cluster with at least two isolates, one to three iso-lates (depending on the cluster size) were selected forMLST. For larger clusters, we selected isolates from boththe centre and the fringe of the Raman cluster. TheMLST sequence type was assigned through the MLSTwebsite (http://www.MLST.net to PubMLST.org). Statistical analysis Chi-square ( v 2 ) test was applied to compare the antibioticresistance rates between MRSA and MSSA isolates aswell as PVL-positive MSSA and PVL-negative MSSA iso-lates using statistical software packages SPSS version 16.0(SPSS Inc., Chicago, IL). This analysis was only per-formed in case resistance was present in 10 or more iso-lates per group. A  P  value less than 0.05 was consideredsignificant. Results Prevalence of MRSA and PVL-positive MSSA A total of 259  S. aureus  were collected consecutively byclinical culture: Denpasar (Sanglah Hospital), 40 strains;Malang (Dr. Saiful Anwar Hospital), 77 strains; Padang(Dr. M. Djamil Hospital), 79 strains; Semarang (Dr. Table 1  Origin (ward/outpatient clinic and specimen) of   S. aureus  isolates collected in the studyWard/outpatient clinicNo. of isolates (%)Blood ( n  =  17) Pus ( n  =  144) Sputum ( n  =  37) Other §  ( n  =  61)Surgery 0 38 (26.4) a,i 1 (2.7) 0Internal medicine 0 21 (14.6) b,j 13 (35.1) e 2 (3.3) q Paediatric 0 5 (3.5) k 0 5 (8.2) r Emergency 1 (5.9) 6 (4.2) l 0 0ICU 5 (29.4) g 2 (1.4) m 5 (13.5) 0Mixed ward* 11 (64.7) h 42 (29.2) c,n 3 (8.1) 11 (18.0) f  Outpatient clinic †  0 30 (20.8) d,o 15 (40.5) p 28 (45.9) s Unidentified ward ‡  0 0 0 15 (24.6)MRSA ( n  =  17) 0 15/144 (10.4) 1/37 (2.7) 1/61 (1.6)PVL-positive MSSA ( n  =  48) 4/17 (23.5) 38/144 (26.4) 2/37 (5.4) 4/61 (6.6)MRSA, methicillin-resistant  Staphylococcus aureus ; MSSA, methicillin-sensitive  S. aureus ; ICU, intensive care unit.*Occupied by adults only (no children) separated between male and female for mixed medical cases (i.e. internal medicine, neurology,surgery). † Consisted of outpatient clinics of : surgery, ear nose and throat, dentistry, internal medicine, pulmonology and dermatology. ‡ Missing ward identity. § Sterile sites (amnion fluid, joint fluid, pleural fluid), eye secretion, ear secretion, nose, throat, urine, tissue and vaginal discharge.MRSA ( n  =  17):  a 5 (29.4%),  b 3 (17.6%),  c 3 (17.6%),  d 3 (17.6%),  e 1 (5.9%),  f  2 (11.8%). PVL-positive MSSA (n = 48):  g 1 (2.1%),  h 3(6.3%),  i 13 (34.2%),  j 4 (8.3%),  k 2 (4.2%),  l 1 (2.1%),  m 1 (2.1%),  n 8 (16.7%),  o 8 (16.7%),  p 2 (4.2%),  q 1 (2.1%),  r 1 (2.1%),  s 3 (6.3%). 612  © 2016 John Wiley & Sons Ltd Tropical Medicine and International Health  volume 21 no 5 pp 610  –  618 may 2016 D. Santosaningsih  et al.  Staphylococcus aureus  in Indonesia

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