The influence of antibiotics on transitory resistome during gut colonization with CTX-M-15 and OXA-162 producing Klebsiella pneumoniae ST15

Abstract

Great efforts have been made to limit the transmission of carbapenemase-producing Enterobacteriaceae (CPE), however, the intestinal reservoir of these strains and its modulation by various antibiotics remain largely unexplored. Our aim was to assess the effects of antibiotic administration (ampicillin, ceftazidime, ciprofloxacin) on the establishment and elimination of intestinal colonization with a CTX-M-15 ESBL and OXA-162 carbapenemase producing Klebsiella pneumoniae ST15 (KP5825) in a murine (C57BL/6 male mice) model. Whole genome sequencing of KP5825 strain was performed on an Illumina MiSeq platform. Conjugation assays were carried out by broth mating method. In colonization experiments, 5 × 10⁶ CFU of KP5825 was administered to the animals by orogastric gavage, and antibiotics were administered in their drinking water for two weeks and were changed every day. The gut colonization rates with KP5825 were assessed by cultivation and qPCR. In each of the stool samples, the gene copy number of bla_OXA-162 and bla_CTX-M-15 were determined by qPCR. Antibiotic concentrations in the stool were determined by high pressure liquid chromatography and a bioanalytical method. The KP5825 contained four different plasmid replicon types, namely IncFII(K), IncL, IncFIB and ColpVC. IncL (containing the bla_OXA-162 resistance gene within a Tn1991.2 genetic element) and IncFII(K) (containing the bla_CTX-M-15 resistance gene) plasmids were successfully conjugated. During ampicillin and ceftazidime treatments, colonization rate of KP5825 increased, while, ciprofloxacin treatments in both concentrations (0.1 g/L and 0.5 g/L) led to significantly decreased colonization rates. The gene copy number bla_OXA-162 correlated with K. pneumoniae in vivo, while a major elevation was observed in the copy number of bla_CTX-M-15 from the first day to the fifteenth day in the 0.5 g/L dose ceftazidime treatment group. Our results demonstrate that commonly used antibiotics may have diverse impacts on the colonization rates of intestinally-carried CPE, in addition to affecting the gene copy number of their resistance genes, thus facilitating their stable persistance and dissemination.