Table 1.
Phenotypic and genotypic characteristics of carbapenem-resistant clinical isolates and transconjugants of Klebsiella pneumoniae, as well as of the wastewater Klebsiella pneumoniae isolates used as recipient cells in the conjugation assays.
Table 2.
Genetic determinants associated with resistance to different classes of antibiotics found in each clinical Klebsiella pneumoniae isolate resistant to meropenem based on whole genome sequence.
Fig 1.
Pulse-Field Gel Electrophoresis (PFGE) of the plasmids of Klebsiella pneumoniae isolates SC.99 (1), E2FC13 (2), KP1-388 (3), KP1-080 (4), KP1-349 (5), KP2-448 (6), KP2-465 (7), KP3-685 (8), and the plasmids of the transconjugants E5: KP1-388-SCC.99 (9), F10: KP1-388-E2FC13 (10), C1: KP1-080-E2FC13 (11), A1: KP1-349-E2FC13 (12), B3: KP2-448-E2FC13 (13), B4: KP2-448-E2FC13 (14), D2: KP2-465-E2FC13 (15).
The orange line represents a sample irrelevant for the study and cropped from the original figure, shown in S1 Fig. In conjugation assays, the strains used as recipient cells were SC.99 and E2FC13 with three plasmids (170 kbp, 70 kbp, 30 kbp) and one plasmid (200 kbp), respectively. The isolates used as donor cells were KP1-388, KP1-080 and KP1-349, all with two plasmids with same sizes (290 kbp and 140 kbp) and KP2-448 and KP2-465, each with four plasmids (160 kbp, 136 kbp, 110 kbp, 55 kbp and 150 kbp, 136 kbp, 110 kbp, 55 kbp, respectively). The isolate KP3-685 has two plasmids (150 kbp, 80 kbp). The transconjugant E5 has four plasmids (170 kbp, 140 kbp, 70 kbp, 30 kbp). The transconjugants F10, C1 and A1 have two plasmids with same sizes (200 kbp, 140 kbp). The transconjugants B3 and D2 have also the plasmid of 200 kbp and a plasmid of 55 kbp. The transconjugant B4 has the plasmid of 200 kbp and a plasmid of 170 kbp. The DNA ladder (M) contains Salmonella enterica serovar Braenderup H9812 (ATCC) 239 digested with XbaI.
Fig 2.
Intensity profiles reflecting the optical maps of the plasmids detected in clinical isolates and found to contain the blaKPC-3 gene.
(a) The 140 kbp plasmid detected in isolates KP1-388, KP1-080 and KP1-349. (b) The 55 kbp plasmid detected in isolates KP2-448 and KP2-465. The open circles in the shaded regions indicate the location of the blaKPC-3 gene. The circle indicating the gene location in KP2-448 in (b) has been shifted vertically for increased visibility.
Fig 3.
Intensity profiles reflecting the optical maps of the plasmids found in isolate KP3-685.
(a) The 150 kbp plasmid present in KP3-685, lacking the blaKPC-3 gene, and the 140 kbp plasmid present in KP1-080 (patient 1), harbouring the blaKPC-3 gene (indicated by the open circle in the shaded region). Shown is the best matching comparison, with a p-value of 0.63 (threshold for similarity set to p-value ≤ 0.01). (b) The 80 kbp plasmid present in KP3-685 and the 136 kbp plasmid present in KP2-448 (patient 2), both lacking the blaKPC-3 gene, and the 140 kbp plasmid present in KP1-080 (patient 1), harbouring the blaKPC-3 gene (indicated by the open circle in the shaded region). The intensity profiles for the 80 kbp plasmid of isolate KP3-685 and 136 kbp plasmid of isolate KP2-488 have been shifted vertically (+1 and -1 unit, respectively) for increased visibility. Note that the intensity profile of the 140 kbp plasmid is shifted relative to the intensity profile shown in Fig 2, due to circular permutation.
Fig 4.
Intensity profiles reflecting the optical maps of the 140 kbp conjugative plasmid harbouring the blaKPC-3 gene.
Conjugative plasmid of 140 kbp present in (a) the clinical isolate KP1-388 and the corresponding transconjugants KP1-388_E2FC13 (F10) and KP1-388_SCC.99 (E5) and (b) the clinical isolate KP1-080 and the corresponding transconjugant KP1-080_E2FC13 (C1) and (c) the clinical isolate KP1-349 and the corresponding transconjugant KP1-349_E2FC13 (A1). The open circles in the shaded regions indicate the location of the blaKPC-3 gene. The circles indicating the gene location in E5 in (a) and C1 in (b) have been shifted vertically for increased visibility.
Fig 5.
Intensity profiles reflecting the optical maps of the 55 kbp conjugative plasmid harbouring the blaKPC-3 gene.
Conjugative plasmid of 55 kbp present in (a) the clinical isolate KP2-448 and the corresponding transconjugant KP2-448_E2FC13 (B3) and (b) the clinical isolate KP2-465 and the corresponding transconjugant KP2-465_E2FC13 (D2). The open circles in the shaded regions indicate the location of the blaKPC-3 gene.
Fig 6.
Intensity profiles reflecting the optical maps of the 170 kbp plasmid present in transconjugant KP2-448_E2FC13 (B4) and the 55 kbp and 136 kbp plasmids present in the Klebsiella pneumoniae clinical isolate KP2-448.
The open circles in the shaded region indicate the blaKPC-3 gene position on the 55 kbp and 170 kbp plasmid, respectively. The intensity profiles for the 55 kbp and 136 kbp plasmids have been shifted vertically (+1 and -1 unit, respectively) for increased visibility.