Download EV0449 ePoster Viewing Resistance mechanisms

EV0449
ePoster Viewing
Resistance mechanisms
Klebsiella pneumoniae co-producing OXA-48 and VIM carbapenemases
Alma Sotillo-Torquemada1, Ana Sarria Visa*1, Guillermo Ruiz-Carrascosa1
1Hospital
Universitario La Paz, Microbiología Y Parasitología, Madrid, Spain
Background: In the last years, infections by carbapenemase-producing Enterobacteriaceae have
caused important hospital outbreaks worldwide. Most outbreaks are produced by singlecarbapenemase producers, but Klebsiella pneumoniae co-producing two different carbapenemases
have been observed.
During the last five years OXA-48-producing K. pneumoniae have spread in Spain. This expansion
has been polyclonal, with several STs involved, but in most cases a single IncL/M plasmid has been
found associated to the OXA-48 carbapenemase gene. In Hospital La Paz (Madrid) VIM metallo-betalactamase-producing Enterobacteriaceae have spread independently of OXA-48 and associated to the
In113 integron. In one case, a Salmonella enterica isolate was found to harbour this integron in the
same IncL/M plasmid thought this did not have an OXA-48 gene.
Recently, a carbapenem-resistant K. pneumoniae isolate was obtained and found to have the OXA-48
and VIM carbapenemase genes. Our aim was to determine whether the two genes were in the same
or different plasmids.
Material/methods: A K. pneumoniae isolate harbouring the OXA-48 carbapenemase and VIM
metallo-β-lactamase genes, and susceptible to rifampicin was isolated in our hospital. The presence of
both carbapenemase genes was detected by qPCR OXVIKP-G® (Progenie Molecular). Identification
was done with MALDI Biotyper (Bruker Daltronik GmbH) and susceptibility testing was done by panel
44 Microscan® (Siemmens) microdilution.
Rifampicin-resistant Escherichia coli BM21 strain was used as the receptor in conjugation
experiments. Transconjugants were selected in LB agar plates containing ceftazidime (4 µg/ml) and
rifampicin (300 µg/ml).
Transconjugant colonies were analyzed by PCR to determine the presence or absence of VIM and/or
OXA-48. The plasmid IncL/M incompatibility group was verified by PCR using the primers:
forward: 5'-GAGTTCCAGAGAGAGTACC-3',
reverse: 5'-GTAAGGTTATGTAAACGCCGC-3'
Results: A K. pneumoniae isolate that carried the OXA-48 and VIM carbapenemase genes was
obtained from sputum sample. The isolate had a meropenem MIC of 8 mg/l (Table 1).
Transconjugants were readily obtained in ceftazidime-rifampicin selection plates. PCR analysis of the
transconjugants, showed that in most cases they contained only the VIM beta-lactamase gene (10/13),
while only in three cases the OXA-48 and VIM genes appeared together and there were not colonies
with OXA-48 gene alone.
Both, the parental strain, and the transconjugants containing the two beta-lactamase genes had an
IncL/M plasmid, while those transconjugants that had only the VIM gene did not, indicating that in this
case the VIM gene is not associated to the IncL/M plasmid.
Conclusions: A carbapenem-resistant K. pneumoniae isolate was obtained that contained OXA-48
and VIM carbapenemase genes. The two genes were located in different plasmids and only the OXA48 gene appeared associated to the IncL/M plasmid.
Table 1: Antibiotic susceptibility of the VIM- OXA producing Klebsiella pneumoniae isolate determined
by the broth microdilution method. R, resistant; I, intermediate; S, susceptible.