pathogens Article Rapid Detection of bla KPC-9 Allele from Clinical Isolates Konstantina Gartzonika 1,2, * ,† , Petros Bozidis 1,† , Ephthalia Priavali 2 and Hercules Sakkas 1,2   Citation: Gartzonika, K.; Bozidis, P.; Priavali, E.; Sakkas, H. Rapid Detection of bla KPC-9 Allele from Clinical Isolates. Pathogens 2021, 10, 487. https://doi.org/10.3390/ pathogens10040487 Academic Editor: Po-Lin Chen Received: 19 March 2021 Accepted: 15 April 2021 Published: 17 April 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Microbiology Department, Faculty of Medicine, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece; pbozidis@uoi.gr (P.B.); isakkas@uoi.gr (H.S.) 2 Microbiology Department, University Hospital of Ioannina, 45110 Ioannina, Greece; epriaval@cc.uoi.gr * Correspondence: kgartzon@uoi.gr; Tel.: +30-265-100-7716 † These authors contributed equally to this study. Abstract: The emergence of Klebsiella pneumoniae carbapenemase (KPC) nosocomial outbreaks re- lated to specific bla KPC gene variants dictates the need for applicable diagnostic methods for allele discrimination. We report here a simple method of bla KPC-9 allele recognition based on a combination of endonuclease digestion analysis and PCR amplification using unique primers. K. pneumoniae isolates carrying the bla KPC gene were tested. Digestion with RsaI restriction endonuclease was found to efficiently differentiate the bla KPC-2 from the bla KPC-9 variants into two distinct groups of digestion patterns named KPC-2-like and KPC-9-like, respectively. An additional procedure, the amplification refractory mutation system (ARMS) method, was applied to identify the variant within the same group. The principles of this procedure could be developed to identify several bla KPC gene variants, as well as monitoring the spread and evolution of specific KPC variants within local geographical regions. Keywords: K. pneumoniae; carbapenemases; bla KPC-2 ; bla KPC-9 ; RsaI; ARMS; outbreak 1. Introduction Klebsiella pneumoniae carbapenemase-possessing K. pneumoniae (KPC-Kp) clinical iso- lates have been implicated in hospital outbreaks in different geographical regions, including Greece [1]. Several amino acid sequence variants of KPC have been described, suggesting the continued evolution of resistance in the KPC-2 enzyme, which is the most prevalent variant worldwide [2]. To date, the nucleotide sequences of 82 KPC gene variants have been deposited in the National Center for Biotechnology Information (NCBI, U.S. National Library of Medicine) databases [3]. The presence of KPC-Kp strains was reported in Greece in 2008 [4], and since then, epidemiological studies conducted in hospitals throughout the country reported K. pneumoniae strains belonging to 11 sequence types [5], harboring mainly the KPC-2 variant [6] and in some sporadic cases the KPC-3 [6] and KPC-23 [7] variants. Only recently were K. pneumoniae strains carrying a different bla KPC gene variant, the KPC-9 variant, identified among KPC-2 producing isolates of an ongoing outbreak taking place in a tertiary hospital in the Northwestern part of Greece [8]. The emergence of KPC nosocomial outbreaks related to these specific bla KPC gene variants within the Greek territory, dictates the need for applicable diagnostic methods for allele discrimination. Prompt and accurate recognition of KPC variants is critical to identify transmission pathways, especially in cases of highly virulent or resistant strains which may require the implementation of specific infection control measures to restrict their dissemination. Although bla KPC gene direct sequencing or whole-genome sequencing (WGS) are the most appropriate methods for K. pneumoniae subtyping, they are both time-consuming procedures, requiring specialized equipment and personnel. We here report a simple method based on a combination of polymerase chain reaction (PCR) amplification using unique primers and endonuclease digestion analysis that could provide a safe shortcut in the positive discrimination of the bla KPC-9 alleles in clinical samples. Pathogens 2021, 10, 487. https://doi.org/10.3390/pathogens10040487 https://www.mdpi.com/journal/pathogens