Summary:
The present study has been focused on two main objectives on the one hand the
detection and characterization of plasmid-mediated AmpC β-lactamases in
Enterobacteriaceae, along with an evaluation of the effectiveness of various
inhibitors in their detection and an assessment of a modified CLSI-ESBL test
and on the other hand the investigation of novel carbapenemase genes which have
recently been introduced to the Greek region.
A total of 105 Enterobacteriaceae isolates (K.pneumoniae, E.coli, K.oxytoca,
P.mirabilis) collected over a 9 year period from 2004 to 2012, exhibiting
reduced susceptibility or resistance either to cefoxitin (FOX) and/or cefotetan
(CTT) along with reduced susceptibility or resistance to at least one extended
spectrum cephalosporin were further investigated for the presence of plasmid
AmpC β-lactamases. Plasmid-mediated AmpCs, ESBLs and the VIM-type
carbapenemases were sought by PCR amplification and subjected to direct
sequencing. Susceptibility testing was performed with disk diffusion and
ertapenem MICs were evaluated with the Etest method. Clonal relationship was
investigated by either enterobacterial repetitive intergenic consensus PCR
(ERIC-PCR) or repetitive sequence based PCR (REP-PCR). Four AmpC inhibitors,
cloxacillin (CLOX), phenyl-boronic acid (PBA), 3-acetyl-phenyl-boronic acid
(APA) and 3-amino-phenyl-boronic (APBA) acid were evaluated, the latter in two
final concentrations (300 and 600 μg/disk) with FOX, CTT, ceftazidime (CAZ) and
cefotaxime (CTX) as substrates. Finally a modified CLSI ESBL test with the use
of the aforementioned inhibitors and CTX and CAZ was evaluated.
Altogether 59 isolates harbored plasmid-mediated AmpCs, with 35 also
co-producing ESBLs. The MOX-2 β-lactamase was identified with the highest
frequency followed by the CMY-2 enzyme. CMY-4, CMY-13 and CMY-31 were also
detected along with three novel to the region enzymes, DHA-1 and two new AmpCs
a CMY-4-like and a MIR-2-like. All P.mirabilis belonged to one clonal type,
K.oxytoca isolates presented two distinct clonal types while K.pneumoniae and
E.coli isolates presented 8 and 11 clonal types respectively.
FOX resistance was found to be a discriminating parameter performing better
than CTT as an initial screening tool. In regards to the confirmatory
phenotypic AmpC disk assay, FOX in combination with APBA produced the best
results, successfully identifying 57/59 isolates. The proposed modified CLSI
ESBL test provided a significantly better overall performance when performed
with CLOX (sensitivity 91,4% and specificity 75%), PBA (100%, 75%) and APBA
(97,1%, 79,1%) in comparison to the standard CLSI ESBL test (85,7%, 58,3%). APA
as an inhibitor failed to enhance the modified CLSI ESBL test exhibiting a
suboptimal sensitivity of 54,3%.
Between January 2010 and June 2013, 132 non-repetitive carbapenem-resistant
Enterobacteriaceae isolates, which gave positive the modified Hodge test (ΜΗΤ)
and were phenotypically suspected of metallo-β-lactamase production (MBL), were
recovered from patients hospitalized in Ioannina University Hospital. Molecular
testing verified the presence in 78 K. pneumoniae isolates, collected from 71
patients, of the blaNDM-1 gene. The blaCTX-M-15, blaOXA-1 and blaTEM-1 genes
were also present in most isolates. The blaNDM-1 gene was located on an IncFII
type plasmid, of c. 95kb, flanked upstream by a non-truncated ISAba125 element
and downstream by the bleMBL gene. Carbapenem resistance was transferable at a
very low rate of ~ 1,3x10-7. Genotyping clustered all K. pneumoniae isolates
into a single clonal type with one subtype and MLST assigned them to sequence
type ST11. Two outbreaks were noted, the first between November-December 2011
involving four patients and the second initiated in May 2012 and ongoing,
involving the remaining patients. All but two cases were characterized as
hospital-acquired. No links to immigration or travel history to endemic areas
were established.
From December 2011-March 2012, 13 ertapenem-resistant K. pneumoniae isolates,
with a positive MHT, and negative phenotypic screening tests for MBL and KPC
production, were recovered from nine patients. All isolates harboured the
blaOXA-48 gene along with blaCTX-M-15 and blaOXA-1 genes. The blaOXA-48 gene
was located on a self-transferable IncL/M-type plasmid of c. 62 kb, which
harboured no other resistance genes. IS1999 was located upstream of blaOXA-48
gene. Genetic disruptions of the OmpK35 and OmpK36 genes associated with
permeability defects were not detected. The isolates belonged to a unique PFGE
clone and MLST assigned them to sequence type ST11. All cases were
characterized as hospital-acquired and none of them was linked to immigration
or had a history of travel in endemic areas.
Over a period of approximately 2,5 years initiated in 2010, three K.pneumoniae
isolates were retrieved form urine samples of a single female patient, of which
the first two with an interval of 41 days, exhibiting resistance to cephamycins
and extended spectrum cephalosporins, with positive screening tests for AmpC β-
lactamase production and a negative modified CLSI-ESBL test. The initial two
isolates exhibited carbapenem resistance, gave a positive ΜΗΤ and negative
screening tests for class A and B carbapenemase production.
All isolates harbored the blaOXA-1 and blaDHA-1 genes and additionally the
first two carried the blaOXA-162 gene. IS1999 was located upstream of
blaOXA-162 gene. Genetic disruptions of the OmpK35 and OmpK36 genes associated
with ertapenem resistance were not detected. Plasmid profiling indicated the
presence of three plasmids of ~140kb, ~62kb and 2kb in the first two while the
latter lacked 62kb plasmid. Conjugation experiments failed to transfer β-lactam
resistance. All K.pneumoniae belonged to a unique PFGE clone and MLST assigned
them to sequence type ST11.
