Multidrug Resistant Klebsiella pneumoniae Sequence Type 258 Producing KPC-2 Carbapenamase

Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria are a prominent cause of multidrug-resistant nosocomial infections, recognized in 2014 by the WHO as a public health threat. Carbapenemases are β-lactamases capable to hydrolyze antibiotics of last resort. In recent years, the bacteria of sequence type 258 (ST258) became the predominant clone in the U.S.A. accounting for over 70% of K. pneumoniae isolates carrying blaKPC carbapenemases. BAA-1705 is a strain of ST258 used by the Clinical Laboratory Standards Institute as a positive control in diagnosis of carbapenemases. Despite its clinical importance little is known about the plasmids it contains and their composition. Mobile genetic elements such as plasmids and transposons are known to provide bacteria with selective advantages such as antibiotic resistance. Therefore, identifying the composition of plasmids in K. pneumoniae BAA-1705 can help understand its evolution and prevent its threat. The objective of this study was to describe the plasmids found in carbapenemase producing K. pneumoniae BAA-1705. We hypothesized that hospital-adapted bacteria accumulate antibiotic resistance genes because they are constantly selected by different types of antibiotics. The bacterial isolate was obtained from American Type Culture Collection. Plasmid DNA was extracted using Qiagen Midikit and sequenced using Illumina. Gaps or ambiguities were closed using PCR with Sanger sequencing. The assembled plasmids were annotated using RAST, followed by manual comparative curation. Our study revealed that BAA-1705 contains multiple plasmids of different sizes (ranging from 5K to 200K base pairs long). The smallest plasmid found does not carry any antibiotic resistant genes or transposases. The next plasmid in size contains a gene that confers resistance to aminoglycoside antibiotics; it also has 2 transposases. We also found a plasmid that confers resistance to aminoglycosides and β-lactams; it contains 6 transposases. Other plasmid contains 3 genes all conferring resistance to β-lactams, and 12 transposases. Lastly, the largest plasmid contains genes that confer resistance to 5 different classes of antibiotics; it has resistance against: chloramphenicol, macrolides, trimethoprim, sulfonamide, and aminoglycosides; it has 37 transposases. Our data showed that the hospital strain BAA-1705 carried multiple antibiotic resistant genes and that the plasmids containing more of such genes also carried more transposases. Our results show that bacteria from clinical setting evolved by accumulating different genes that confer resistance to the same class of antibiotics. This can be due to the transposases facilitating the exchange of genetic material. Altogether, our study adds new DNA sequencing data on K. pneumoniae BAA-1705.