Purpose: In recent years, multidrug-resistant Acinetobacter baumannii has appeared and caused outbreaks of hospital infections all over the world. Close monitoring of this pathogen and other A. baumanii complex species is considered of critical importance to public health organizations. The reliable identification method able to distinguish A. baumannii from genetically close Acinetobacter species is needed, because these species are unable to be differentiated by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect A. baumanii complex species, and Acinetobacter lwoffii which is frequently detected from the human specimens, and to investigate the distribution of these organisms in dental hospital using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S ribosomal RNA (16S rRNA) gene and DNA gyrase subunit B (gyrB) of each species of A. baumanii complex species. Swab samples were collected from ten dental spittoon units in dental hospital, and the distribution of A. baumanii complex species was investigated using a multiplex PCR. Results: These primers were able to distinguish each species of A. baumanii complex species clearly. A. baumanii and A. calcoaceticus were detected at 20.0% and 10.0% in ten swab samples, respectively. On the other hand, A. nosocomialis, A. lowffii, and A. pittii were detected from no sample. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.
References
[1]
Espinal, P., Seifert, H., Dijkshoorn, L., Vila, J. and Roca, I. (2012) Rapid and Accurate Identification of Genomic Species from the Acinetobacter baumannii (Ab) Group by MALDI-TOF MS. Clinical Microbiology and Infection, 18, 1097-1103. https://doi.org/10.1111/j.1469-0691.2011.03696.x
[2]
Toh, B.E.W., Paterson, D.L., Kamolvit, W., Zowawi, H., Kvaskoff, D., Sidjabat, H., Wailan, A., Peleg, A.Y. and Huber, C.A. (2015) Species Identification within Acinetobacter calcoaceticus-baumannii Complex Using MALDI-TOF MS. Journal of Microbiological Methods, 118, 128-132. https://doi.org/10.1016/j.mimet.2015.09.006
[3]
Hsueh, P.R., Kuo, L.C., Chang, T.C., Lee, T.F., Teng, S.H., Chuang, Y.C., Teng, L.J. and Sheng, W.H. (2014) Evaluation of the Bruker Biotyper Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry System for Identification of Blood Isolates of Acinetobacter Species. Journal of Clinical Microbiology, 52, 3095-3100. https://doi.org/10.1128/JCM.01233-14
[4]
Sousa, C., Botelho, J., Silva, L., Grosso, F., Nemec, A., Lopes, J. and Peixe, L. (2014) MALDI-TOF MS and Chemometric Based Identification of the Acinetobacter calcoaceticus-Acinetobacter baumannii Complex Species. International Journal of Medical Microbiology, 304, 669-677. https://doi.org/10.1016/j.ijmm.2014.04.014
[5]
Wisplinghoff, H., Paulus, T., Lugenheim, M., Stefanik, D., Higgins, P.G., Edmond, M.B., Wenzel, R.P. and Seifert, H. (2012) Nosocomial Bloodstream Infections Due to Acinetobacter baumannii, Acinetobacter pittii and Acinetobacter nosocomialis in the United States. The Journal of Infection, 64, 282-290. https://doi.org/10.1016/j.jinf.2011.12.008
[6]
Schleicher, X., Higgins, P.G., Wisplinghoff, H., Korber-Irrgang, B., Kresken, M. and Seifert, H. (2013) Molecular Epidemiology of Acinetobacter baumannii and Acinetobacter nosocomialis in Germany over a 5-Year Period (2005-2009). Clinical Microbiology and Infection, 19, 737-742. https://doi.org/10.1111/1469-0691.12026
[7]
Wang, X., Chen, T., Yu, R., Lü, X. and Zong, Z. (2013) Acinetobacter pittii and Acinetobacter nosocomialis among Clinical Isolates of the Acinetobacter calcoaceticus-baumannii Complex in Sichuan, China. Diagnostic Microbiology and Infectious Disease, 76, 392-395. https://doi.org/10.1016/j.diagmicrobio.2013.03.020
[8]
Chuang, Y.C., Sheng, W.H., Lauderdale, T.L., Li, S.Y., Wang, J.T., Chen, Y.C. and Chang, S.C. (2014) Molecular Epidemiology, Antimicrobial Susceptibility and Carbapenemase Resistance Determinants among Acinetobacter baumannii Clinical Isolates in Taiwan. Journal of Microbiology, Immunology, and Infection, 47, 324-332. https://doi.org/10.1016/j.jmii.2013.03.008
[9]
Fukatsu, A., Tsuzukibashi, O., Suzuki, H., Asaka, K., Ono, Y., Fuchigami, M., Kobayashi, T., Uchibori, S., Takahashi, Y., Komine, C., Konishi, Y., Ogura, Y., Omori, H., Wakami, M., Murakami, H. and Fukumoto, M. (2021) One-Step Multiplex PCR for Simultaneous Detection and Identification of Eight Medically Important Candida Species. Open Journal of Stomatology, 11, 14-24. https://doi.org/10.4236/ojst.2021.111002
Higgins, P.G., Lehmann, M., Wisplinghoff, H. and Seifert, H. (2010) gyrB Multiplex PCR to Differentiate between Acinetobacter calcoaceticus and Acinetobacter Genomic Species 3. Journal of Clinical Microbiology, 48, 4592-4594. https://doi.org/10.1128/JCM.01765-10
[12]
Bernards, A.T., van der Toorn, J., van Boven, C.P.A. and Dijkshoorn, L. (1996) Evaluation of the Ability of a Commercial System to Identify Acinetobacter Genomic Species. European Journal of Clinical Microbiology and Infectious Diseases, 15, 303-308. https://doi.org/10.1007/BF01695662
[13]
Gerner-Smidt, P., Tjernberg, I. and Ursing, J. (1991) Reliability of Phenotypic Tests for Identification of Acinetobacter Species. Journal of Clinical Microbiology, 29, 277-282. https://doi.org/10.1128/jcm.29.2.277-282.1991
[14]
Dijkshoorn, L., Nemec, A. and Seifert, H. (2007) An Increasing Threat in Hospitals: Multidrug-Resistant Acinetobacter baumannii. Nature Reviews Microbiology, 5, 939-951. https://doi.org/10.1038/nrmicro1789
[15]
Horrevorts, A., Bergman, K., Kollee, L., Breuker, I., Tjernberg, I. and Dijkshoorn, L. (1995) Clinical and Epidemiologic Investigations of Acinetobacter Genomospecies-3 in a Neonatal Intensive-Care Unit. Journal of Clinical Microbiology, 33, 1567-1572. https://doi.org/10.1128/jcm.33.6.1567-1572.1995
[16]
Poirier, S., Rue, O., Peguilhan, R., Coeuret, G., Zagorec, M., Champomier-Verges, M.C., Loux, V. and Chaillou, S. (2018) Deciphering Intra-Species Bacterial Diversity of Meat and Seafood Spoilage Microbiota Using gyrB Amplicon Sequencing: A Comparative Analysis with 16S rDNA V3-V4 Amplicon Sequencing. PLOS ONE, 13, e0204629. https://doi.org/10.1371/journal.pone.0204629
[17]
Roux, S., Enault, F., Bronner, G. and Debroas, D. (2011) Comparison of 16S rRNA and Protein-Coding Genes as Molecular Markers for Assessing Microbial Diversity (Bacteria and Archaea) in Ecosystems. FEMS Microbiology Ecology, 78, 617-628. https://doi.org/10.1111/j.1574-6941.2011.01190.x
[18]
Ghebremedhin, B., Layer, F., Konig, W. and Konig, B. (2008) Genetic Classification and Distinguishing of Staphylococcus Species Based on Different Partial Gap, 16S rRNA, hsp60, rpoB, sodA, and tuf Gene Sequences. Journal of Clinical Microbiology, 46, 1019-1025. https://doi.org/10.1128/JCM.02058-07
[19]
Links, M.G., Dumonceaux, T.J., Hemmingsen, S.M. and Hill, J.E. (2012) The Chaperonin-60 Universal Target Is a Barcode for Bacteria That Enables De Novo Assembly of Metagenomic Sequence Data. PLOS ONE, 7, e49755. https://doi.org/10.1371/journal.pone.0049755
[20]
Adekambi, T., Drancourt, M. and Raoult, D. (2009) The rpoB Gene as a Tool for Clinical Microbiologists. Trends in Microbiology, 17, 37-45. https://doi.org/10.1016/j.tim.2008.09.008
[21]
Rosenthal, S.L. (1974) Sources of Pseudomonas and Acinetobacter Species Found in Human Culture Materials. American Journal of Clinical Pathology, 62, 807-811. https://doi.org/10.1093/ajcp/62.6.807
