1.MI714-Leong Sui Sien Available online at http://jurnal.permi.or.id/index.php/mionline DOI: 10.5454/mi.7.4.1ISSN 1978-3477, eISSN 2087-8575 Vol 7, No 4, November 2013, p 137-143 *Corresponding author; Phone/Fax: +60-82-581-388, Email: leongsuisien87@gmail.com Abuse of antibiotic use is becoming a major concern of today’s society. It is also the major cause of antibiotic resistance in clinical practice. Since the discovery of penicillin in the 1940s, the emergence of antibiotic resistance has been highlighted. According to Dorsch (2007), the excessive use of antibacterials in agriculture and clinical therapy has led to the phenomenon of antibiotic resistance. Antibiotic resistan- ce decreases our ability in treating infections and diseases, affecting the proper infection control, and prevention strategies (Dorsch, 2007). Antibiotic resistant bacteria have also widely spread in the environment. Transmission of resistant gene in these pathogenic bacteria may cause health problem to human. According to Varaldo (2002), Streptococcus pyogenes has become resistant to erythromycin and able to enter human respiratory cells. Staphylococcus aureus has also been found to be resistant to a variety of antibiotics (Akano et al. 2009). Thus, the studies of There is a growing concern on the occurrence of antimicrobial resistance. Development of multiple antibiotic resistant bacteria has overtaken new drug development and threatened the patients with untreatable infections. This study was conducted to isolate and characterize the antibiotic resistant bacteria from swiftlet farm houses located in various places including Kota Samarahan, Semarang, Saratok, Betong, Sarikei, Sibu, Sepinang, Maludam, Miri, and Kuching in Sarawak, Malaysia. Five feces samples were collected randomly from each site. One gram of the feces sample was diluted in 9 mL of 0.85% normal saline solution. The diluted sample was plated on Trypticase Soy agar plates and incubated at 37±1 °C for 24 h. A total of 500 bacteria isolates were then identified using 16S rRNA analysis method. Disc diffusion method was then used to confirm the resistant phenotypes of these isolates. The results showed that the means of the bacterial colony count were significantly -1 different (p<0.05) from one another, with the highest log CFU g (9.22±0.72) found in Kota Samarahan and the 10 -1 lowest log CFU g (6.03±0.62) in Betong. Besides, the isolated bacteria were identified as 96% Gram positive 10 bacteria and 4% Gram negative bacteria. The isolated bacteria were highly resistant to penicillin G (36.80±23.87%), ampicillin (28.60±17.13%), and rifampicin (16.90±13.70%). Thus, swiftlet feces are good reservoir for a range of antibiotic resistant bacteria which may pose a potential health hazard to human. Key words: antibiotic resistance, bacteria, isolation, swiftlet feces Dewasa ini, tingkat kekhawatiran pada munculnya resistensi terhadap antimikroba semakin meningkat. Laju kemunculan bakteri dengan resistensi ganda terhadap antibiotik lebih tinggi dibandingkan dengan penemuan obat baru, sehingga membahayakan pasien-pasien dengan infeksi yang tidak tertangani. Penelitian ini ditujukan untuk mengisolasi bakteri yang resisten terhadap antibiotik dari peternakan walet (swiftlet) yang tersebar di Kota Samarahan, Semarang, Saratok, Betong, Sarikei, Sibu, Sepinang, Maludam, Miri, dan Kuching di Sarawak, Malaysia dan mengkarakterisasinya. Lima sampel feses diambil secara acak dari masing-masing situs. Satu gram sampel fees didilusi dalam 9 mL 0.85% larutan salin normal. Sample terdilusi kemudian di sebar pada media agar Tripticase Soy dan diinkubasi pada 37±1 °C selama 24 jam. Lima ratus isolat bakteria kemudian diidentifikasi berdasarkan urutan 16S rRNA. Resistensi dibuktikan menggunakan metode cakram difusi (disk diffusion method). Hasil penelitian ini menunjukkan bahwa nilai rata-rata penghitungan koloni bakteri berbeda secara -1 signifikan (p<0.05) antara isolat satu dengan lainnya. Nilai log CFU g tertinggi ditemukan di Kota Samarahan 10 (9.22±0.72) sementara nilai terendah ditemukan di Betong (6.03±0.62). Identifikasi juga menunjukkan bahwa 96% isolat adalah bakteri gram positif sementara hanya 4% bakteri gram negatif. Bakteri terisolasi menunjukkan resistensi tinggi terhadap Penisilin G (36.80±23.87%), Ampisilin (28.60±17.13%), dan Rifampisin (16.90±13.70%). Maka dapat disimpulkan bahwa feses walet merupakan sumber bakteri yang resisten antibiotik, sehingga boleh jadi berpotensi berbahaya terhadap manusia. Kata kuci: bakteri,feses walet, isolasi, resistensi antibiotik Isolation and Characterization of Antibiotic Resistant Bacteria from Swiftlet Feces in Swiftlet Farm Houses in Sarawak, Malaysia 1 1 2 LEONG SUI SIEN *, SAMUEL LIHAN , LING TECK YEE , 1 3 CHIA HWA CHUAN , AND LIM CHAN KOON 1 2 Department of Molecular Biology; Department of Chemistry; 3 Department of Zoology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Kota Samarahan 94300, Sarawak, Malaysia antibiotic resistance are as vital as providing optimum treatment for patients (Dorsch 2007) and predicting the emerging of resistant pathogens (Allen et al. 2010). Swiftlet feces are usually used as organic fertilizer in agricultural industry because it is rich in nutrient, water, nitrogen, phosphorus, potassium, and minerals (Nyakundi and Mwangi 2011). Most bacteria are able to grow in the swiftlet feces. Organic fertilizers containing these antibiotic resistant bacteria may contaminate vegetables. Improper washing of the contaminated vegetables may also spoil our foods in the kitchen, causing harmful diseases to human. Intensive and extensive abuse of antibiotics usage have caused the formation of an antibiotic resistant genes pool in the environment. Thus, there is a growing public health concern on the microbiological risk of antibiotic resistance of these bacteria in the swiftlet feces to human health. Therefore, this study was conducted to identify and characterize the antibiotic resistant bacteria present in the swiftlet farm houses. MATERIALS AND METHODS Locations of the Study Areas. Swiftlet houses were situated at Kota Samarahan, Semarang, Saratok, Betong, Sarikei, Sibu, Sepinang, Maludam, Miri, and Kuching in Sarawak, Malaysia. Sample Collection. Sampling and sample processing procedures for the isolation of bacteria from the swiftlet feces were carried out as described by Nyakundi and Mwangi (2011). Five samples were collected randomly from the floor of the sampling sites in each swiftlet house. The samples were collected using the spatula and then transferred into sterile bag for storage and then further analysed in the laboratory. Sample was diluted by mixing 1 g of dropping in 9 mL of sterile 0.85% saline solutio\n. The diluted sample was plated on Trypticase Soy agar (Scharlau, Spain) plates and incubated at 37±1 °C for 24 h. The number of -1 bacterial colony forming unit (CFU g ) was calculated according to Nyakundi and Mwangi (2011) as the number of colony per plate multiplied by the dilution factor. Five to 10 colonies were randomly picked for further identification. DNA Extraction. The bacterial DNA was extracted by boiling method described by Maria et al. (2008) with minor modification. The bacterial cultures were prepared by growing the bacteria in Luria broth (Scharlau, Spain) at 37 ± 1°C for 24 h. One thousand and 500 µL of overnight bacterial culture was transferred into 1.5 mL microcentrifuge tube and centrifuged at 10 138 SIEN ET AL. Microbiol Indones 000 rpm for 5 min. The supernatant was then discarded. Five hundred microliters of sterile distilled water was added and vortexed to resuspend the cell pellet. The content of the microcentrifuge tubes were then boiled for 10 min together with the tubes and then immediately placed on ice for 5 min. After 5 min, the microcentrifuge tubes were centrifuged at 10 000 rpm for 10 min and the supernatant was collected. DNA Sequencing Polymerase Chain Reaction Analysis. The isolated bacteria were genotypically characterized using 16S rRNA identification method with minor modification (Woo et al. 2000; Kumar et al. 2009). Primers 27F (5’-AGAGTTTGATC CTGGCTCA G-3’) and 519R (5’-GWATTACCGCGG CKGCTG-3’) were used. PCR was performed in reaction mixtures containing 20 µL of DNA, 1.0 µL of each 20 pmol primers (First Base), 3 µL of 10 mM deoxynucleoside triphosphates mix (Promega), 6.0 µL of 25 mM MgCl (Promega), 10.0 µL of 5x Buffer 2 solution (Promega), 8.0µL of distilled water, and 1µL of Taq polymerase (Promega). PCR was performed for 35 cycles with initial denaturation at 95 °C for 10 min, denaturation at 94 °C for 30 sec, annealing at 55 °C for 1 min, primer extension 72 °C for 1.5 min, and final extension at 72 °C for 10 min. The PCR products were visualized by ethidium bromide staining after agarose gel electrophoresis for 30 min at 90 volt. Sequencing Analysis. DNA purification was carried out using QIAGEN sequencing purifying kit (QIAquick Gel Extraction Kit, USA). Purified DNA product was sent for DNA sequencing at First BASE Laboratories Sdn. Bhd. in Selangor, Malaysia. All the DNA sequences were compared using BLAST program to determine the closest matches of the identities. Disc Diffusion Method. Antibiotic susceptibility testing was conducted using disc diffusion method according to the method described by National Committee for Clinical Laboratory Standards (NCCLS) (2012) and Zulkifli et al. (2009). Antibiotic discs (Oxoid, England) used for testing represents agents historically or currently used in clinical practice. Escherichia coli ATCC 25922 was used as positive control. Among the antibiotic discs used in this testing were chloramphenicol (30 µg), ampicillin (10 µg), tetracycline (30 µg), streptomycin (10 µg), gentamycin (10 µg), erythromycin (15 µg), cephalothin (30 µg), nitrofurantoin (300 µg), tobramycin (10 µg), rifampicin (5µg), kanamycin (30 g), sulphamethoxazo- le/trimethoprim (1.25/ 23.75 µg), amikacin (30 µg), imipenem (10 µg), ceftriaxone (30 µg), penicillin G (10 U), doxycycline (30 µg), ceftazidime (30 µg), norflaxacin (10 µg), vancomycin (30 µg), piperacillin (100 µg), Ciprofloxacin (5 µg), and Nalidixic acid (30 µg). The medium used in this study was Mueller- Hinton agar (MHA). A sterile cotton swab was dipped into a bacterial suspension with turbidity 0.5 McFarland and used to inoculate the surface of the MHA plate evenly, using different bacteria for each plate. The plate was allowed to dry for 5 min. A pair of sterile forceps was used to pick up the antibiotic disc and placed on the surface of the MHA agar plate. The plate was incubated at 37±1 °C for 24 h. The diameter of inhibition zone was measured and the results were recorded as sensitive (S) or resistance (R) base on WHO Drug Information and NCCLS (Yah et al. 2007). Statistical Analysis. Statistical analysis of the data was conducted using SPSS Statistics version 21.0 program. Besides, all the data were analyzed for analysis of variance (ANOVA). All differences between the means were compared using Turkey multiple range test after a significant F-test at P<0.05. RESULTS Bacterial Colony Count. Table 1 showed that the -1 means of the bacterial colony counts (log CFU g ) 10 were significantly different (F=8.265, P<0.05) from -1 one sampling site to another The highest log CFU g10 of 9.22±0.72 was found in Kota Samarahan and -1 the lowest log CFU g of 6.03±0.62 in Betong. The 10 mean bacterial colony counts collected from Kota Samarahan, Maludam, Miri, Kuching, and Sibu were significantly higher than those from other sampling sites such as Saratok, Betong, Semarang, Sepinang, and Sarikei. Identification of Bacteria in Swiftlet Feces. A total of 500 bacterial isolates were obtained from Kota Samarahan, Saratok, Betong, Maludam, Miri, Kuching, Semarang, Sepinang, Sarikei, and Sibu. The results showed that a total of 480 (96%) bacterial isolates were identified as Gram positive, while only 20 (4%) were identified as Gram negative bacteria. The distribution of the bacterial isolates in the swiftlet feces from the different sampling sites is shown in Fig 1. Although all these bacterial isolates were found in all the sites, Staphylococcus sp. was the most highly prevalent bacteria, followed by Bacillus sp., Lysinibacillus sp., Enterococcus harae, Sporosarcina sp., E. coli, Paenibacillus sp., Pseudomonas aeruginosa, Leucobac-ter iarius, and Dermacoccus sp. Antibiotic Susceptibility Test. A total of 500 bacterial isolates were identified and disc diffusion method was then used to confirm the resistant phenotypes of these isolates. The percentages of resistance to 23 types of antibiotics among bacteria isolated from Kota Samarahan, Saratok, Betong, Maludam, Miri, Kuching, Semarang, Sepinang, Sarikei, and Sibu was shown in Tables 2. The highest frequencies of antibiotic resistance were detected against penicillin G (36.80±23.87%), followed by ampicillin (28.60±17.13%), and rifampicin (16.90±13. Volume 7, 2013 Microbiol Indones 139 Site -1 Mean of bacterial colony counts (log CFU g )10 Kota Samarahan Saratok Betong Maludam Miri Kuching Semarang Sepinang Sarikei Sibu Total 9.22 ± 0.72 a* 6.23 ± 0.93 b 6.03 ± 0.62 b 7.47 ± 0.49 abcd 8.76 ± 0.14 ad 8.38 ± 0.16 acd 7.14 ± 0.50 bcd 6.64 ± 0.83 bc 6.95 ± 1.68 bc 7.52 ± 1.01 abcd 7.43 ± 1.26 Table 1 The means of colony counts of bacteria isolated from Kota Samarahan, Saratok, Betong, Maludam, Miri, Kuching, Semarang, Sepinang, Sarikei, and Sibu in Sarawak, Malaysia bacteria were capable to grow both aerobically and anaerobically. Besides, Kota Samarahan is situated at the coastal area which has sufficiently nutrient-rich water considering the shallow coastal depth water- level and the abundance of sunlight in this are, thus more conducive for bacterial growth. The present bacterial count was higher than the findings reported by Nyakundi and Mwangi (2011). In their report, the 6 total bacterial viable count was between 2.4 x 10 and 6 -1 7.8 x 10 CFU g from Marabou stock (Leptoptilos crumeniferus) droppings. The results of this microbiological examination on the bacterial distribution in the samples of swiftlet feces randomly collected at Kota Samarahan, Semarang, Saratok, Betong, Sarikei, Sibu, Sepinang, Maludam, Miri, and Kuching in Sarawak, Malaysia (Fig 1) revealed that most of these bacterial isolates were Gram positive bacteria. Staphylococcus sp. was the most prevalent, followed by Bacillus sp., Lysinibacillus sp. B4, E. harae, Sporosarcina sp., E. coli, Paenibacillus sp., P. aeruginosa, L. iarius, and Dermacoccus sp. Staphylococcus sp. and Bacillus sp. were highly prevalent bacteria mainly because they are facultative anaerobic bacteria capable of growing both aerobically 70%). The bacteria isolated from different sites showed different levels of resistance toward the antibiotics (Table 2). The bacteria have developed resistance to at least one antibiotic. The analysis of variance test showed that the levels of the resistance toward 23 tested antibiotics were significantly different (F=6.780, P<0.05) from one another. The mean resistance levels toward penicillin G and ampicillin were significantly higher (P <0.05) than the other tested antibiotics such as chloramphenicol, tetracycline, streptomycin, gentamy- cin, erythromycin, cephalothin, nitrofurantoin, tobra- mycin, rifampicin, kanamycin, sulphamethoxazole/tri- methoprim, amikacin, imipenem, ceftriaxone, doxycy- cline, ceftazidime, norflaxacin, vancomycin, piperacil- lin, ciprofloxacin, and nalidixic acid. DISCUSSION Bacterial colony count was performed to determine the total number of microorganisms present in the collected swiftlet feces samples. The log of mean 10 bacterial colony count per g was the highest (9.22±0.72) in samples collected from Kota Samarahan (Table 1), mostly because these facultative anaerobic Microbiol Indones140 SIEN ET AL. Fig 1 Swiftlet feces bacterial distributions (%) in Kota Samarahan, Semarang, Saratok, Betong, Sarikei, Sibu, Sepinang, Maludam, Miri, and Kuching in Sarawak, Malaysia. 0 20 40 60 80 100 120 D is tr ib u ti o n o f b ac te ri a (% ) Sampling sites Leucobacter iarius strain 40 Proteus sp. Pseudomonas aeruginosa strain 123 Dermacoccus sp. 103 Paenibacillus sp. Gh-134 Sporosarcina sp. Enterococcus harae strain ss33b Escherichia coli Staphylococcus sp. Lysinibacillus sp. B4 Bacillus sp. Se m ar an g K ot a Sa m ar ah an Sa ra to k B et on g Sa rik ei Si bu Se pi na ng M al ud am M iri K uc hi ng Volume 7, 2013 Microbiol Indones 141` A n ti b io ti c R e si st a n c e r a te ( % ) M e a n K o ta S a m a ra h a n (n = 5 0 ) S a ra to k (n = 5 0 ) B e to n g (n = 5 0 ) M a lu d a m (n = 5 0 ) M ir i (n = 5 0 ) K u c h in g (n = 5 0 ) S e m a ra n g (n = 5 0 ) S e p in a n g (n = 5 0 ) S a ri k e i (n = 5 0 ) S ib u (n = 5 0 ) P e n ic il li n A m p ic il li n 4 2 6 2 3 8 1 0 2 8 4 2 8 2 0 1 6 2 0 2 8 .6 0 ± 1 7 .1 3 a b * P e n ic il li n G 4 0 6 6 7 2 1 4 4 6 6 4 1 2 2 0 1 6 1 8 3 6 .8 0 ± 2 3 .8 7 a P ip e ra c il li n 4 8 0 0 0 0 4 0 0 0 1 .6 0 ± 2 .8 0 c C e p h a lo sp o r in C e ft ri a x o n e 4 2 8 0 4 0 2 2 0 6 0 6 .4 0 ± 1 2 .8 2 c C e ft a z id im e 3 8 1 6 1 2 6 0 2 4 2 1 8 1 8 2 1 5 .1 1 ± 1 5 .5 0 b c C e p h a lo th in 4 4 2 0 4 4 1 8 4 1 6 1 2 4 0 1 2 .6 0 ± 1 3 .0 7 b c P h e n ic o l te tr a c y c li n e C h lo ra m p h e n ic o l 0 8 0 0 1 8 0 2 0 0 0 2 .8 0 ± 5 .9 0 c Q u in o lo n e s N o rf lo x a c in 4 1 6 0 0 0 0 0 0 0 0 3 .4 5 ± 6 .8 2 c N a li d ix ic a c id 4 8 0 0 0 0 0 0 0 0 1 .2 0 ± 2 .7 0 c F lu ro q u in o lo n e C ip ro fl o x a c in 4 1 6 4 0 4 2 0 0 0 0 3 .0 0 ± 4 .9 2 c M a c ro li d e E ry th ro m y c in 0 3 0 0 2 4 1 0 2 0 8 0 5 .6 0 ± 9 .2 8 c A m in o g ly c o si d e G e n ta m ic in 5 4 2 0 4 0 0 0 2 2 0 1 8 0 1 1 .8 0 ± 1 7 .2 2 b c K a n a m y c in 2 8 0 0 0 2 2 0 0 0 0 3 .2 0 ± 7 .0 7 c A m ik a c in 0 8 0 0 0 0 8 0 0 0 1 .6 0 ± 3 .3 7 c T o b ra m y c in 0 8 0 0 0 2 2 0 0 0 0 3 .0 0 ± 7 .1 3 c C a r b a p e n e m Im ip e n e m 0 8 0 0 0 0 0 0 0 0 0 .8 0 ± 2 .5 3 c N it ro fu r a n N it ro fu ra n to in 0 1 6 0 0 1 8 2 2 2 0 0 0 5 .8 0 ± 9 .0 2 c A n sa m y c in R if a m p ic in 4 4 2 8 4 6 2 4 2 6 2 0 9 6 2 1 6 .9 0 ± 1 3 .7 0 b c S tr e p to m y c in 4 8 0 0 0 0 1 0 0 0 0 2 .2 0 ± 3 .8 2 c T ab le 2 R es is ta n ce t o 2 3 t y p es o f an ti b io ti cs a m o n g b ac te ri a is o la te d f ro m s w if tl et f ec es c o ll ec te d a t K o ta S am ar ah an , S ar at o k , B et o n g , M al u d am , M ir i, K u ch in g , S em ar an g , S ep in an g , S ar ik ei , a n d S ib u in S ar aw ak , M al ay si a * m ea n s w it h t h e sa m e su p er sc ri p ts a re n o t si g n if ic an tl y d if fe re n t at 5 % l ev el . In conclusion, the mean of bacteria colony count -1 (log CFU g ) was the highest in Kota Samarahan and 10 the lowest in Betong. There were more Gram positive than Gram negative bacteria isolated from the swiftlet feces in Sarawak, Malaysia. Staphylococcus sp. and Bacillus sp. were the most highly prevalent bacteria in the swiftlet farm houses. The isolated bacteria showed high resistance to penicillin G and ampicillin when tested against 23 types of antibiotics commonly used in current medical practice. If the antibiotic resistant bacteria cannot be controlled or killed by antibiotics, it might become a serious threat to public health. ACKNOWLEDGMET This research was funded partially by Fundamental Research Grant Scheme under the grant No. FRGS/01(1 6)/745/2010(31). REFERENCES Akano SO, Daini OA, Ojo MO, Smith SI, Akinside KA. 2009. Comparative analysis of antibiotic resistance and R-plasmid of Staphylococcus aureus isolates from human and dog samples. Afri J Clin Exp Microbiol. 10(3):136-143. Allen HK, Donato J, Wang HH, Cloud-Hansen KA, Davies J, Handelsman J. 2010. Call of the wild: antibiotic resistance genes in natural environments. Nat Rev Microbiol. 8:251-259. doi:10.1038/nrmicro2312. Bozdogan BU, Esel D, Whitener C, Browne FA, Appelbaum PC. 2003. Antibacterial susceptibility of a vancomycin-resistant Staphylococcus aureus strain isolated at Hershey medical Center. J Antimicrob Chemoth. 52(5):864. doi:10.1093/jac/dkg457. Chih MY, Ming FL, Chung HL, Yi TH, Chia TH, Ming LL. 2009. Characterization of antimicrobial resistance patterns and integrons in human fecal Escherichia coli in Taiwan. Jpn J Infect. 62(3):177-181. Cirz RT, Chin JK, Andes DR, de Crecy-Lagard V, Craig WA, Romesberg FE. 2005. Inhibition of mutation and combating the evolution of antibiotic resistance. PLoS Biol. 3(6): e176. doi:10.1371/journal.pbio.0030176. Clements LD, Miller BS, Streips UN. 2002. Comparative growth analysis of the facultative anaerobes Bacillus subtilis, Bacillus licheniformis, and Escherichia coli. US National Library of Medicine National Institute of health. 25(2):284-286. Dorsch MR. 2007. Rapid Detection of Bacterial Antibiotic Resistance: Preliminary evaluation of PCR assays targeting tetracycline resistance genes. Human Protection and Performance Division 2007. Enne VI, Delsol AA, Roe JM, Bennett PM. 2004. Rifampicin resistance and its fitness cost in Enterococ- cus faecium. J Antimicrob Chemoth. 53(2):203-207. and anaerobically (Clements et al. 2002). Besides, Bacillus sp. was able to produce endospores that can stay dormant under stressful environment for a long period of time (Wayne et al. 2000), thus able to survive even in dry swiftlet feces. Contamination of bird feed in the feces sample could also be the reason for the occurrence of Staphylococcus sp. According to Kocijan et al. (2009), Staphylococcus sp. was ubiquitous and commonly found in bird feed. According to Simpson (2002), it is normal to find E. coli in swiftlet feces samples because almost all wild birds have E. coli as their normal flora. This study showed that E. coli found in the swiftlet feces samples was normal because almost all wild birds contain E. coli (Simpson, 2002). Nyakundi and Mwangi (2011) and Literak et al. (2007) had isolated E. coli from the Marabou Stock (Leptoptilos crumeniferus) droppings and rooks droppings samples. The results, as shown in Table 2, have revealed that most of the bacteria were resistant to penicillin G and ampicillin antibiotics. According to Li and Nikadio (2009), bacteria exhibit an enzymatic deactivation mechanism by producing β-lactamases. In my study, it was shown that Staphylococcus sp. was the most highly prevalent bacteria occurring in all the swiftlet houses. Bozdogan et al. (2003) reported that Staphylococc- us sp. had higher antibiotic resistance tendency toward penicillin, methicillin, tetracycline, and erythromycin. Besides, Cirz et al. (2005) showed the similar result showing that Enterococcus sp. had higher resistance towards rifampicin. Acquired resistance such as mutation of genes encoding the bacterial protein (Cirz et al. 2005; Enne et al. 2003) occur and caused antibiotic resistance in most bacteria. Cirz et al. (2005) also mentioned that the occurrence of vancomycin resistant Enterococcus was high and a lot of researcher had shown the data as early as 1987. Pseudomonas sp. was another highly prevalent pathogen and antibiotic resistance bacteria (Poole 2004). Pseudomonas sp. exhibits multiple antibiotic resistance mechanisms by developing multidrug efflux pumps encoded by antibiotic resistant genes and bacterial cellular envelopes with lower permeability to antibiotics (Poole 2004). Most of the E. coli were highly resistant to piperacillin and streptomycin (Wittwer et al. 2005). Streptomycin has been the first-line antimicrobial and widely used as growth promoters in human and agricultural field in Taiwan (Chih et al. 2009). Similar result was shown by Chih et al. (2009) stated that extensive usage of streptomycin antibiotics caused resistance in E. coli. Microbiol Indones142 SIEN ET AL. Volume 7, 2013 Microbiol Indones 143 Simpson VR. 2002. Wild animals as reservoirs of infectious diseases in the UK. Vet J. 163(2):128-146. doi:10.1053/ tvjl.2001.0662. Varaldo P. 2002. Antimicrobial resistance and susceptibility testing: an evergreen topic. H, Henry JM, Peter S. 2000. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol. 64(3):548-572. doi:10.1128/MMBR.64.3.548- 572.2000. Wayne LN, Nobuo M, Gerda H, Henry JM, Peter s. 2000. Resistance of Bacillus endospore to extreme terrestrial and extraterrestrial environment. Microbiol Mol Biol Rev. 64(3):548-572. doi:10.1128/MMBR.64.3.548- 572.2000. Wittwer M, Keller T, Wassenaar TM, Stephen R, Howald D, Regula G, Bissig-Choisat B. 2005. Genetic diversity and antibiotic resistance patterns in Campylobacter population isolated from poultry farm in Switzerland. Appl Environ Microbiol. 71(6):2840-2847. doi:10.1128 /AEM.71.6.2840-2847.2005. Woo PCY, Leung PKL, Leung KW, Yuen KY. 2000. Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species from a bone marrow transplant recipient. Molecular Pathology. 53(4):211- 215. doi:10.1136/mp.53.4.211. Yah CS, Chineye HU, Eghafona NO. 2007. Multi-antibiotics- resistance plasmid profile of enteric pathogens in pediatric patients from Nigeria. Biokemistri. 19(1):35- 42. Zulkifli Y, Alitheen NB, Son R, Raha AR, Samuel L. 2009. Random amplified polymorphic DNA-PCR and ERIC PCR analysis on Vibrio parahaemolyticus isolated from cockles in Padang, Indonesia. Int Food Res J. 16:141-150. doi:10.1093/jac/dkh044. Kocijan IE, Prukner-Radovcic, Beck R, Galov A, Marinculic A, Susic G. 2009. Microflora and internal parasites of the digestive tract of Eurasian friffon vultures (Gyps fulvulus) in Croatia. Eur J Wildl Res. 55(1):71-74. doi:10.1007/s10344-008-0209-4. Kumar P, Ramakritinan CM, Kumaraguru AK. 2009. 16S rRNA based identification of Aeromonas sp. kumar by constructing phylogenetic tree and identification of regulatory elements from the harmful red tide bloom, Gulf of Mannar. Int J Oceans Oceanogr. 3(2):29-35. Li X, Nikadio H. 2009. Efflux-mediated drug resistance in bacteria. An Update. Drug 69(12):1555-1623. doi:10.21 65/11317030-000000000-00000. Literak I, Vanko R, Dolejska M, Cizek A, Karpyskova R. 2007. Antibiotic resistant Escherichia coli and Salmonella in Russian rooks (Corvus frugilegus) intering in Czech Republic. Lett Appl Microbial. 45(6):616-621. doi:10.1111/j.1472-765X.2007.02236.x. Maria IQO, Juan DDC, Manuel M, Maria JB, Pilar M. 2008. Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in Real- Time PCR for serum samples from patient s with Brucellosis. American Society for Microbiology. 15(2):293-296. National Committee for Laboratory Standards (NCCLS). 2012. Performance standards for antimicrobial susceptibility testing; twenty-second informational supplement. In Approved standard M100-S22. Volume 32(3).Villanova, Pa: United States. Nyakundi WO and Mwangi W. 2011. Isolation and Characterisation of pathogenic bacteria and fungi from Leptoptilos crumeriferu (Marabau Strok) dropping. J Appl Sci Tech Environ Sanit. 1(1):93-103. Poole K. 2004. Efflux-mediated multiresistance in Gram- negative bacteria. Clin Microbiol Infect. 10(1):12-26. doi:10.1111/j.1469-0691.2004.00763.x. 1: 137 2: 138 3: 139 4: 140 5: 141 6: 142 7: 143