Ultrastructural Changes in Clinical and Microbiota Isolates of Klebsiella pneumoniae Carriers of Genes bla SHV, bla TEM, bla CTX-M, or bla KPC When Subject to β-Lactam Antibiotics

Abstract

The aim of this study was to characterize the ultrastructural effects caused by β-lactam antibiotics in Klebsiella pneumoniae isolates. Three K. pneumoniae clinical isolates were selected for the study with resistance profiles for third-generation cephalosporins, aztreonam, and/or imipenem and with different resistance genes for extended-spectrum β-lactamases (ESBL) or Klebsiella pneumoniae carbapenemase (KPC). Two K. pneumoniae isolates obtained from the microbiota, which were both resistant to amoxicillin and ampicillin, were also analyzed. In accordance with the susceptibility profile, the clinical isolates were subjected to subminimum inhibitory concentrations (sub-MICs) of cefotaxime, ceftazidime, aztreonam, and imipenem and the isolates from the microbiota to ampicillin and amoxicillin, for analysis by means of scanning and transmission electron microscopy. The K. pneumoniae isolates showed different morphological and ultrastructural changes after subjection to β-lactams tested at different concentrations, such as cell filamentation, loss of cytoplasmic material, and deformation of dividing septa. Our results demonstrate that K. pneumoniae isolates harboring different genes that encode for β-lactamases show cell alterations when subjected to different β-lactam antibiotics, thus suggesting that they possess residual activity in vitro, despite the phenotypic resistance presented in the isolates analyzed.

Figure 1

(a–e) Transmission electron micrographs of isolate K3C from K. pneumoniae. (a) bacterial cell without treatment (control)—preserved morphology, cytoplasmic membrane (arrow), cell wall (w), and cytoplasm (C) intact. (b-c) Cells subjected to CTX (64 μg mL⁻¹)—decreased cytoplasmic material (C), presence of membrane compartments (stars), electron-lucent spaces (asterisks) and disorganization of the membrane and cell wall. (d) Cell subjected to CAZ (32 μg mL⁻¹)—elongated aspect morphology with loss of cytoplasmic material (C) and the presence of small membrane compartments (stars). (e) Cell subjected to ATM (32 μg mL⁻¹)—normal morphology in the presence of electron-lucent spaces (asterisks) in the cytoplasm (C). Bars = 0.5 μm. CAZ: ceftazidime; CTX: cefotaxime; and ATM: aztreonam.

Figure 2

(a–h) Transmission electron micrographs of isolate K16R from K. pneumoniae. (a) Untreated bacterial cell—preserved morphology, cytoplasmic membrane (arrow), cell wall (w), and cytoplasm (C) intact. (b) Cell subjected to CTX (64 μg mL⁻¹)—presence of large electron-lucent space due to increased periplasmic space (stars) and reduced cytoplasmic material (C). (c–e) Cells subjected to CAZ (16 μg mL⁻¹). (c-d) Filamentous cells and the presence of membrane compartments containing cytoplasmic material (star). (e) Elongated cell showing loss of cytoplasmic material (star). (f–h) Cell subjected to ATM (4 μg mL⁻¹)—loss of cytoplasmic material (C) and presence of membrane compartments (stars). Bars = 0.5 μm. CAZ: ceftazidime; CTX: cefotaxime; and ATM: aztreonam.

Figure 3

(a–h) Transmission electron micrographs of isolate K652 of K. pneumoniae. (a) Untreated bacterial cell—cell wall with preserved morphology (w) and cytoplasm (C) intact, besides regular septation (arrowheads). (b-c) Cell subjected to CTX (98 μg mL⁻¹)—presence of cells not grown and rounded (stars) at the ends of cells with normal morphology. (d–f) Cells subjected to CAZ (27 μg mL⁻¹)—consecutive formation of septa (arrowheads) providing elongation of bacterial morphology. Observe disorganization of membrane and cell wall with elastic aspect. (g-h) Cell subjected to IMP (16 μg mL⁻¹)—morphology of elongated appearance with formation of consecutive septa (arrowheads). Bars = 0.5 μm. CAZ: ceftazidime; CTX: cefotaxime; and IMP: imipenem.

Figure 4

(a–i) Scanning electron micrographs of isolate K652. (a-b) Control cells with preserved morphology. (c-d) Cells subjected to CTX (98 μg mL⁻¹). Observe spherical cells that are not grown being formed from cells with preserved morphology. (e–g) Cells subjected to CAZ (27 μg mL⁻¹). Observe elongated cells forming bacterial filaments. (h-i) Cells subjected to IMP (16 μg mL⁻¹). Observe elongated cells due to unfinished successive divisions. Bars = 2 μm. CAZ: ceftazidime; CTX: cefotaxime; and IMP: imipenem.

Figure 5

(a–d) Scanning electron micrographs of isolate K16R. (a) Control cells with preserved morphology. (b) Cells subjected to CTX (32 μg mL⁻¹). (c) Cells subjected to CAZ (16 μg mL⁻¹). (d) Cells subjected to ATM (4 μg mL⁻¹). Bars = 2 μm. CAZ: ceftazidime; CTX: cefotaxime; and ATM: aztreonam.

Figure 6

(a–d) Scanning electron micrographs of isolate K3C. (a) Control cells with preserved morphology. (b) Cells subjected to CTX (32 μg mL⁻¹). (c) Cells subjected to CAZ (32 μg mL⁻¹). (d) Cells subjected to ATM (32 μg mL⁻¹). Bars = 2 μm. CAZ: ceftazidime; CTX: cefotaxime; and ATM: aztreonam.

Figure 7

(a–c) Transmission electron micrographs of isolate K21.1-F and K58.1-F of K. pneumoniae. (a) Isolate K21.1-F submitted to 0.5 μg mL⁻¹ ampicillin. Observe irregular formation of septa in a dividing cell (arrow). (b) Isolate K58.1-F subjected to 0.5 μg mL⁻¹ of ampicillin. Cells present electron-lucent (fine arrow), and electron-dense spaces with different sizes and shape throughout the bacterial cytoplasm (arrowheads), in addition to reduction of cytoplasmic contents (star). (c) Isolate K58.1-F subjected to 4 μg mL⁻¹ of amoxicillin. The cells exhibit altered morphologies with cell elongation (large arrow) and undefined forms (asterisk), with the presence of electron-lucent structures with shape and varying sizes scattered throughout the cell cytoplasm (fine arrow). Bars = 0.5 μm.