الملخص الإنجليزي
Background: Antimicrobial resistance mediated by AmpC β-lactamases, are a
significant cause of public health concern. Tracing the genotypic and phenotypic
characterization of the prevalent AmpC β-lactamase producing bacteria is essential.
This knowledge aids in making informed empirical decisions. These enzymes confer
resistance to a wide range of β-lactam antibiotics except carbapenems, leading to their
gross overuse. Assessment of carbapenem sparing protocols like newer β-lactam/β lactamase inhibitors - ceftazidime/avibactam (CZA) and meropenem/vaborbactam
(MVA) as well as combinations of older drugs are useful exercises. These can guide
the clinicians to prescribe appropriate carbapenem sparing antimicrobials.
Aim and Objectives: To assess six phenotypic (three commercial and three in-house)
tests for detection of AmpC. To characterize antimicrobial resistance and virulence
genes in AmpC producing Gram negative bacteria by whole genome sequencing
(WGS). To assess efficacy of CZA and MVA and identify carbapenem sparing
combinations among amikacin (AK), levofloxacin (LV), and piperacillin/tazobactam
(TZP).
Methods: A total of 116 non-duplicate Gram negative bacteria were screened by
cefoxitin (FOX) disc and 82 FOX resistant of K. pneumoniae (n=25), E. coli (n=22),
P. aeruginosa (n=17), E. cloacae (n=6), K. aerogenes (n=4), S. marcescens (n=4) and
C. freundii (n=4) were isolated from Sultan Qaboos University Hospital (SQUH) and
investigated for AmpC β-lactamase production by six methods. The conventional
AmpC tris-EDTA and disc approximation method and the commercial D69C AmpC
detection set, D72C ESBL, AmpC and carbapenemase detection set, combined disc
for AmpC and ESBL and AmpC- MIC strips were evaluated for detection of AmpC.
Ten representative isolates were whole genome sequenced and analysed through
bioinformatic online tools for multilocus sequence type (MLST), acquired resistance
genes, plasmids and virulence factors. In addition, CZA and MVA MICs were
analysed against 31 isolates by E strips. Antimicrobial synergy between AK-LV, AK TZP, TZP-LV was assessed in 11 isolates by the E-test cross method.
Results: The prevalence of AmpC- β-lactamases was 16% at SQUH. E. coli was a
major carrier of plasmid mediated AmpC (26.5%) followed by Klebsiella pneumoniae
(23.4%). Phenotypically, 61% of AmpC were detected by tris-EDTA, 76% by disc
approximation, 75% by D69C AmpC detection set, 74% by D72C AmpC, ESBL and
carbapenemase detection set, 76% by combination disc and 63% by AmpC-MIC
strips. Most sensitive and specific phenotypic assays were the combination disc test
with 96% sensitivity and 94% specificity and disc approximation test with 94%
sensitivity and 75% specificity while cefoxitin screening was less sensitive (75%) with
poor specificity 24%. WGS revealed DHA-1 as the predominant AmpC gene. New
MLST were detected in both E. coli (ST-96 and ST-12742) and Klebsiella pneumoniae
(ST-3157 and ST-5118). All the 4 E. coli produced both AmpC and ESBLs. CTX-M 15 was the dominant ESBL gene. Both E. coli and K. pneumoniae carried a large
battery of virulence genes, of which adhesion afa and fim predominated. CZA and
MVA showed excellent in-vitro activity in all AmpC (n=31) tested isolates. The MICs
ranged from 0.032-0.19 μg/ml. AK-PTZ combination revealed good synergy against
K. pneumoniae isolates.
Conclusions: We recommend the in-house disc approximation test and the
commercial combination disc test as excellent tools for detection of AmpC, the former
being easy to interpret, cheaper and easily adopted in most microbiology laboratories.
Cefoxitin test overcalls AmpC and cannot be considered a good stand-alone test for
AmpC detection. DHA-1 was the predominant AmpC gene and MLST analysis
revealed new circulating clones in this region. We recommend E- test cross method
for rapid detection of synergy, thus tailoring antimicrobial combinations to individual
patients. Amikacin-piperacillin/tazobactam combination yielded synergy in K.
pneumoniae. Such combinations allow us to preserve drugs like CZA and MVA for
more difficult to treat infections.
