Gold Nanostar-based Point-of-Care Diagnostics of Carbapenemase-Producing Enterobacteriacae

The ever-increasing spread of antibiotic resistance poses a serious threat to global public health. With the existence of Carbapenem-resistant Enterobacteriaceae (CRE), it renders the use of carbapenems, the last-resort class... [ view full abstract ]

The ever-increasing spread of antibiotic resistance poses a serious threat to global public health. With the existence of Carbapenem-resistant Enterobacteriaceae (CRE), it renders the use of carbapenems, the last-resort class of β-lactam antibiotics, useless against combating against bacterial infections. Such infections reduce the ability to treat complex infections due to the lack of antibiotic options for treatment, leading to CRE-associated mortalities. Current methods of detection, like CarbaNP test and Modified Hodge’s Test, have significant limitations that the time taken for detection of carbapenemase activity ranges between hours to days, and are non-specific in phenotypic profiling, making it challenging to isolate patients rapidly and devise appropriate treatment for infected patients. We propose a methodology by utilizing Surface Enhanced Raman Spectroscopy (SERS) to study bacterial β-lactamase activity. This is done via the use of gold nanostars (AuNS), which have reported excellent SERS properties, conjugated with a β-lactam antibiotic ceftriaxole, as a proof-of-concept study to analyse the changes in the SERS spectra associated with cleavage of the β-lactam ring upon interaction with the New Delhi Metalloproteinase (NDM)-producing Escherichia coli. 

Methods

1) Synthesis of AuNS
The synthesis of AuNS was done using the one-pot seedless protocol, involving 3 reagents: gold (III) chloride, silver nitrate & ascorbic acid (AA), which have been optimised and reported for maximum SERS enhancement signal. 

2) Conjugation of Ceftriaxole onto AuNS
100uL of 5mM CTX solution was added to 100uL of 50pM washed AuNS solution, and incubated for 30 minutes at room temperature. Colloidal stability of the conjugate was tested via addition of 100uL of 0.5X PBS, where the optical properties of the conjugates was characterised using UV-Vis Spectroscopy.

3) Bacterial Studies
30uL of NDM- and non-NDM-producing E. coli suspension was added to cuvettes containing 200uL of AuNS-CTX solution separately. SERS spectra was acquired every 5 minutes up to 45 minutes.

Results & Discussion: SERS Study on β-lactam cleavage
We are able to obtain detection of carbapenemase activity within 25 minutes, with the associated changes in SERS spectra being diminishing of SERS peaks at 1358cm^-1 and 1495cm^-1.