Biochemical identification and molecular characterization of Klebsiella pneumoniae isolated from street foods and drinks in Yogyakarta, Indonesia using 16S rRNA gene




Abstract. Budiarso TY, Amarantini C, Pakpahan S. 2021. Biochemical identification and molecular characterization of Klebsiella pneumoniae isolated from street foods and drinks in Yogyakarta, Indonesia using 16S rRNA gene. Biodiversitas 22: 5452-5458. Yogyakarta is best known as a student city in Indonesia. However, the fact that students spend almost full day in the school gave rise to the sale of a great variety of foods and drinks in its surrounding streets. Unfortunately, most street vendors do not pay attention to food hygiene. This study aims to identify the biochemical and molecular isolates of Klebsiella pneumoniae isolated from street foods and drinks in Yogyakarta using 16S rRNA gene. Data were collected from 120 samples of street foods and drinks sold at schools and several public places. The samples were enumerated using CCA medium to isolate suspected Klebsiella spp. colonies, then physiologically screened and identified using the API-20E kit. Positive isolates were isolated and amplified using the 16S rRNA gene marker for sequencing, which was then compared with the GenBank database to establish molecular identity. The results of biochemical identification obtained 11 isolates confirmed as K. pneumoniae and 1 isolate as K. oxytoca. The phylogenetic analysis showed that only 10 isolates were determined as K. pneumoniae with the reference sequences of K. pneumoniae from infected patients' respiratory and urinary tract. Furthermore, 2 isolates identified as Citrobacter sp. and Micrococcus sp., which are contaminated with Klebsiella spp. were highly found in street foods and drinks. Therefore, hygiene and sanitation need to be implemented when processing and serving these foods.


Al-Agha AGM, Al-Khafaji NJM, Al-Azawi AKS. 2017. Isolation and identification of Klebsiella pneumoniae using API-20E analytical system and conventional PCR assay. Int J Curr Microbiol App Sci 6(8): 203-210.
Alimi BA. 2016. Risk factors in street food practices in developing countries: A review. Food Sci Hum Wellness 5: 141-148.
Alsanie WF. 2020. Molecular diversity and profile analysis of virulence-associated genes in some Klebsiella pneumoniae isolates. Practical Laboratory Medicine, e00152. DOI:10.1016/j.plabm.2020.e00152.
Bagley ST. 1985. Habitat association of Klebsiella species. Infect Control 6(2):52-58. doi:10.1017/s0195941700062603
Bengoechea JA, Pessoa SA. 2019. Klebsiella pneumoniae infection biology: living to counteract host defences. FEMS Mic Rev 43 (2): 123-144.
Caneiras C, Lito L, Mayoralas-Alises S, Díaz-Lobat S, Melo-Cristino J, Duarte A. 2019. Virulence and resistance determinants of Klebsiella pneumoniae isolated from a Portuguese tertiary university hospital centre over a 31-year period. Enfermedades infecciosas microbiologia clinica 37(6): 387-393.
Cappuccino JG, Welsh C. 2017. Microbiology A Laboratory Manual. Pearson Education Limited. England.
Chandran A, Mazumder A. 2015. Pathogenic potential, genetic diversity and population structure of Escherichia coli strains isolated from a forest-dominated watershed (comox lake) in British Columbia, Canada. Appl Environ Microbiol 81(5): 1788-1798.
Farmer JJ, Brenner, DJ. 2015. Obesumbacterium. Bergey’s Manual of Systematics of Archaea and Bacteria, 1–14. DOI:10.1002/9781118960608.gbm01156.
Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39(4): 783-791.
Fuenzalida MJ, Ruegg PL. 2019. Negatively controlled, randomized clinical trial to evaluate intramammary treatment of nonsevere, gram-negative clinical mastitis. Jour of Dairy Sci. DOI:10.3168/jds.2018-16156
Ghosh S, Bandyopadhyay PK. 2019. Molecular characterization of newly identified Klebsiella PKBSG14 and analysis of its effect on immune response and cell cycle progression using common catfish (Channa punctatus) as a model. Microbial Pathogenesis 127: 368-379.
Guo Y, Zhou H, Qin, L, Pang Z, Qin T, Ren H et al. 2016. Frequency, antimicrobial resistance and genetic diversity of Klebsiella pneumoniae in food samples. PLOS ONE 11(4), e0153561. DOI:10.1371/journal.pone.0153561.
Hamza E, Dorgham SM, and Hamza DA. 2016. Carbapenemase-producing Klebsiella pneumoniae in broiler poultry farming in Egypt. Jour of Glob Antimic Res 7: 8-10.
Harada T, Taguchi M, Kawahara R, Kanki M, Kawatsu K. 2018. Prevalence and antimicrobial susceptibility of bacterial pathogens in ready-to-eat foods retailed in Osaka Prefecture, Japan. Journal of Food Protection 81(9): 1450–1458. DOI:10.4315/0362-028x.jfp-18-035
Hassan MM, Belal ESB. 2016. Antibiotic resistance and virulence genes in enterococcus strains isolated from different hospitals in Saudi Arabia. Biotechnology & Biotechnological Equipment 30(4): 726-732.
Ibrahim IA. 2016. 16S rRNA gene sequencing for identification of some enterobacteriaceae species isolated from tigris river. Al-Mus Jour of Sci 27(3): 13-16.
Kim HS, Chon JW, Kim YJ, Kim DH, Kim M and Seo KH. 2015. Prevalence and characterization of extended-spectrum-?-lactamase-producing Escherichia coli and Klebsiella pneumoniae in ready-to-eat vegetables. Inter Jour of Food Mic 207: 83-86
Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences, J. Mol. Evol 16 :111-120.
Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Bio and Evo 33(7): 1870-1874. DOI:10.1093/molbev/msw054 .
Langoni H,, Guiduce et al. 2015. Research of Klebsiella pneumoniae in dairy herds. Pesq. Vet. Bras. 35(1):9-12,
DOI: 10.1590/S0100-736X2015000100003
Maisonneuve E, Cateau E, Delouche M, Quellard N, Rodier MH. 2017. An observational study of phagocytes and Klebsiella pneumoniae relationships: different behaviors. Mic and Infec 19(4-5): 259-266.
Maheux AF, Bouchard S, Bérubé E, Bergeron MG. 2017. Comparison of MI, Chromocult® coliform, and compass CC chromogenic culture-based methods to detect Escherichia coli and total coliforms in water using 16S rRNA sequencing for colony identification. J Water Health 15 (3): 353-359
Onuoha SC, Okafor CO, Aduo BC. 2016. Antibiotic and heavy metal tolerance of bacterial pathogens isolated from agricultural soil. World Jour of Med Sci 13(4): 236-241.
Prabhurajeshwar C, Chandrakanth K. 2019. Evaluation of antimicrobial properties and their substances against pathogenic bacteria in vitro by probiotic Lactobacilli strains isolated from commercial yoghurt. Clin Nut Exp 23: 97-115.
Ramaditya NA, Tono K, Suarjana IGK, Besung, INK. 2018. Isolation Klebsiella sp. at Bali cattle based on level of maturity and breeding location and the pattern of sensitivity against antibacterial. BulVet Udayana 10(1): 26-32.
Richardson LA. 2017. Understanding and overcoming antibiotic resistance. PLOS Biology |
Rowbotham RF, Ruegg, PL. 2016. Bacterial counts on teat skin and in new sand, recycled sand, and recycled manure solids used as bedding in freestalls. Journal of Dairy Science 99(8): 6594–6608. doi:10.3168/jds.2015-10674 .
Saini, M., Raj, A., Bhattacharyya, S., Banik, A., & Chattopadhyay, U. K. 2016. Prevalence of Klebsiella oxytoca in milk samples of metropolitan city Kolkata: A study. Eastern Journal of Medical Sciences, 5(1), 16-18.
Saitou N, Nei M. 1987.The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Bio and Evo 4(4): 406-425,
Salazar, J. K., Carstens, C. K., Ramachandran, P., Shazer, A. G., Narula, S. S., Reed, E., … Schill, K. M. (2018). Metagenomics of pasteurized and unpasteurized gouda cheese using targeted 16S rDNA sequencing. BMC Microbiology, 18(1). doi:10.1186/s12866-018-1323-4
Shiningeni D, Chimwamurombe P, Shilangale R, Misihairabgwi J. 2018. Prevalence of pathogenic bacteria in street vended ready-to-eat meats in Windhoek, Namibia. Meat Science. doi:10.1016/j.meatsci.2018.05.014
Teramura H, Sota K, Iwasaki M, and Ogihara H. 2017. Comparison of the quantitative dry culture methods with both conventional media and most probable number method for the enumeration of coliforms and Escherichia coli /coliforms in food. Let in App Mic 65(1) : 57-65
Zhang S, Yang G, Ye Q, Wu Q, Zhang J, Huang Y. 2018. Phenotypic and Genotypic Characterization of Klebsiella pneumoniae Isolated From Retail Foods in China. Frontiers in Microbiology, 9. doi:10.3389/fmicb.2018.00289.