EXTENDED SPECTRUM Β-LACTAMASE BLACTX-M-3 IN A PLASMID OF A KLEBSIELLA PNEUMONIAE

Plasmids are mobile genetic elements that disseminate antibiotic resistance (AR) genes among bacteria. Extended spectrum β-lactamase (ESBL) genes confer resistance to multiple β-lactam antibiotics including third generation cephalosporins. ESBL genes are typically spread through ESBL carrying plasmids. Klebsiella pneumoniae, an Enterobacteriaceae, is responsible for 8% of all nosocomial infections in the US. K. pneumoniae is known to cause respiratory infections, such as pneumonia - an illness with a 50% mortality rate. The objective of this study is to describe the complete sequence of an ESBL containing plasmid and its genetic neighborhood. An ESBL containing K. pneumoniae strain was identified from a sample collection via MALDI-TOF. The ESBL phenotype was inferred by double disk susceptibility test through the size difference between the zones of inhibition of ceftazidime and ceftazidime/clavulanic acid. A gel of plasmid DNA extraction indicated the presence of a single plasmid with a size of ∼ 140 kbp. This plasmid was completely sequenced. Conjugative experiments were conducted to confirm the mobility of the plasmid between species. The plasmid belonged to IncF incompatibility type and had a size of 129,868 bp. Fifteen genes conferring resistance to 9 groups of antibiotics and one antimicrobial were found : rifampicin(arr3), aminoglycoside (aac(6’)lb-cr, strB, aacC2, aadA16, aph(3’’)-lb), quinolone (qnrS1, aac(6’)lb-cr), macrolide (mphA), sulphonamide (sul1,sul2), trimethoprim (dfrA27), tetracycline (tetA), phenicol (floR), β-lactam (blaTEM-1B, blaCTX-M-3) and quaternary ammonium compounds (qacEdelta1) . There are five tnpA26 transposases within the region containing all the AR genes in the plasmid. The whole AR region of the plasmid is flanked by tnpA26 genes. The ESBL encoding gene, blaCTX-M-3, was found between tnpA26 transposases along with a broad spectrum β-lactamase gene, blaTEM-1B. The same section also contained the antibiotic resistance genes aacC2 and floR, as well as the transposases tnpA and tnpR. Conjugation between our donor strain SW2954 and the recipient strain J53 was successful, indicating the plasmid is conjugative. Plasmid analyses of the environmental K. pneumoniae isolate show that the ESBL encoding gene, blaCTX-M-3, is contained within a transposable element. Furthermore, neighboring regions of blaCTX-M-3, also delimited by transposases, carry additional AR genes, so that the whole AR region of the plasmid forms a composite transposable element. However comparative analysis did not show the whole AR region being mobilized to other plasmids; instead, its constituent parts are being disseminated and shuffled in different bacteria. Comparative phylogenetic analysis of ∼80 kbp of the core of the plasmids shows that rearrangements within the AR region occur at a rapid rate. Our results show that dissemination of blaCTX-M-3 will often go simultaneously with other AR genes, which are part of the same mobile element.