Detection of Extended-Spectrum β-Lactamase and Carbapenemase Activity in Gram-Negative Bacilli Using Liquid Chromatography – Tandem Mass Spectrometry

Figures & data

Table 1 Strain Description with Phenotypic Confirmation of ESBL and Carbapenemase Production

	Strains	ESBL Test Result	MHT/CDDM Result
Reference strains	Klebsiella pneumoniae ATCC BAA-1705	+	+/KPC
	Klebsiella pneumoniae ATCC 700,603	+	NA
	Escherichia coli ATCC 25,922	NA	NA
Isolated strains	Klebsiella pneumoniae (1)	-	+/KPC
	Klebsiella pneumoniae (2)	+	+/KPC
	Providencia stuartii	-	+/OXA 48
	Acinetobacter baumannii	-	+/OXA 48
	Escherichia coli (1)	+	NA
	Escherichia coli (2)	+	NA
	Escherichia coli (3)	+	NA

Abbreviations: ESBL, extended spectrum β-lactamase; MHT, modified Hodge test; CDDM, combined disk diffusion method; ATCC, American Type Culture Collection; AMP, ampicillin; CAZ, ceftazidime; CRO, ceftriaxone; FEP, cefepime; IMI, imipenem; MEM, meropenem; MHT, modified Hodge test; NA, not applicable.

Table 2 The Optimized MRM Transitions for Each Antibiotic

Compound	Transition (m/z)
Ampicillin	350.15→106.25 350.15→160.25
Imipenem	300.15→98.20 300.15→142.20
Meropenem	384.20→141.30 384.20→68.25
Ceftriaxone	555.25→396.15 555.25→125.25
Cefepime	481.25→86.35 481.25→396.15
Ceftazidime	547.30→468.10 547.30→167.30

Abbreviations: MRM, multiple reaction monitoring; m/z, mass to charge.

Table 3 Accuracy, Precision, Retention Time, LODs and LOQs for Each Antibiotic

Compound	Accuracy – RSD (%) *	Precision – RSD (%) **	RT Min	RT RSD%	LOD ng/mL	LOQ ng/mL
Ampicillin	1.6	4.4	2.6	0.6	0.3	1
Imipenem	0.3	6.2	2	0.1	0.1	0.2
Meropenem	3.9	5.4	2.3	0.2	0.1	0.2
Ceftriaxone	2	11.9	2.7	0.2	2.4	7.2
Cefepime	4.2	6.9	2.1	0.4	0.2	0.6
Ceftazidime	5.7	14.6	2.4	0.1	0.2	0.5

Notes: *Determined by ten repeated injections of each calibration standards, **Assessed by repeating the hydrolysis test five times using E coli ATCC 2592.

Abbreviations: RT, retention time; RSD, relative standard deviation; LOD, limit of detection; LOQ, limit of quantification.

Figure 1 MRM chromatograms of mixture of ampicillin (5 µg/mL), imipenem (0.5 µg/mL), meropenem (0.5 µg/mL), ceftriaxone (0.5 µg/mL), cefepime (0.5 µg/mL), and ceftazidime (0.5 µg/mL) dissolved in phosphate buffer.

Table 4 LC-MS/MS Antibiotic Hydrolysis Assay Compared with the Minimum Inhibitory Concentration Test. For Antibiotic Hydrolysis Assay, The Strains Were Incubated for 2 h at 37°C with a Mixture of Antibiotics

Strains	Test	AMP	IMI	MEM	CRO	FEP	CAZ
K. pneumoniae ATCC BAA-1705	LC-MS/MS results*	5.7/R	3.3/R	1.4/R	2.5/R	3.9/R	27.5/R
	MIC results**	˃128/R	8/R	˃ 16/R	64/R	16/R	≥ 64/R
K. pneumoniae ATCC 700,603	LC-MS/MS results*	5.4/R	90.4/S	92.2/S	35.3/R	98.6/S	39.5/R
	MIC results**	˃ 128/R	0.25/S	0.25/S	16/R	2/S	≥ 64/R
E. coli ATCC 25,922	LC-MS/MS results***	100	100	100	100	100	100
	MIC results**	8/S	0.25/S	0.06/S	0.12/S	0.12/S	0.5/S
K. pneumoniae (1)	LC-MS/MS results*	0.9/R	55.6/R	39.9/R	38.4/R	71.9/R	62.5/R
	MIC results**	≥32/R	≥ 16/R	≥ 16/R	8/R	16/R	≥ 64/R
K. pneumoniae (2)	LC-MS/MS results*	0.5/R	0.1/R	0.3/R	0.3/R	92.1/S	0.5/R
	MIC results**	≥ 32/R	8/R	4/R	≥ 64/R	8/SDD	16/R
P. stuartii****	LC-MS/MS results*	N/A	0/R	0/R	9.9/R	22.4/R	19.1/R
	MIC results**	N/A	≥ 16/R	˃ 16/R	≥ 64/R	16/R	≥ 64/R
A. baumannii****	LC-MS/MS results*	N/A	0/R	21.3/R	14.6/R	43.5/R	29.3/R
	MIC results**	N/A	≥ 16/R	≥ 16/R	≥ 64/R	≥ 64/R	≥ 64/R
E. coli (1)	LC-MS/MS results*	42/R	93.2/S	103.1/S	21.1/R	63.3/R	87.6/S
	MIC results**	≥ 32/R	≤ 0.25/S	≤ 0.25/S	≥ 64/R	≥ 64/R	8 I
E. coli (2)	LC-MS/MS results*	22.6/R	92.5/S	90.2/S	27.8/R	52/R	34.5/R
	MIC results**	˃128/R	≤ 0.25/S	≤ 0.25/S	≥ 64/R	16/R	≥ 64/R
E. coli (3)	LC-MS/MS results*	12/R	95/S	91/S	25/R	54/R	37/R
	MIC results**	≥128/R	≤ 0.25/S	≤ 0.25/S	≥ 64/R	16/R	≥ 32/R

Notes: *Results expressed as a percentage of unhydrolyzed antibiotic, hydrolysis is considered present where more than 10% of antibiotic is hydrolyzed for AMP, IMI, MEM and FEP; and 15% for CRO and CAZ. Concentrations of antibiotics: 5 mg/mL for AMP and 0.5 mg/mL for IMI, MEM, CRO, FEP, CAZ. **Results expressed as minimum inhibitory concentration of antibiotic (µg/mL) that can inhibit the growth of bacteria. Susceptibility or resistance were defined according to CLSI breakpoints. For all reference strains MIC corresponded to CLSI qualitative control ranges. ***Strain sensitive to all antibiotics and used as negative control, percentage of unhydrolyzed antibiotic is 100. ****Providencia and Acinetobacter are intrinsically resistant to ampicillin.²⁰ Therefore, in these cases, values of hydrolysis and MC assays for ampicillin do not have clinical utility.

Abbreviations: R, resistant; S, sensitive; I, intermediate; SSD, susceptible dose dependent; AMP, ampicillin; IMI, imipenem; MEM, meropenem; CRO, ceftriaxone; FEP, cefepime; CAZ, ceftazidime, N/A, not applicable.

Figure 2 Hydrolysis level of each antibiotic for Klebsiella pneumoniae ATCC BAA-1705, Acinetobacter baumannii and Escherichia coli.

Note: * Results expressed in percentage of unhydrolyzed antibiotic

Abbreviations: AMP, ampicillin; IMI, imipenem; MEM, meropenem; CRO, ceftriaxone; FEP, cefepime; CAZ,ceftazidime.

Table 5 Advantages and Disadvantages of Minimum Inhibitory Concentration Test and LC-MS/MS-Based Antibiotic Hydrolysis Assay

	The Minimum Inhibitory Concentration Test (MIC)	LC-MS/MS-Based Antibiotic Hydrolysis Assay
Advantages	It tests several antibiotics consecutively Initial equipment costs is low Confidently determine intermediate resistance	It tests several antibiotics consecutively 3 to 5 h for completing the test Low cost of consumables
Disadvantages	High cost of consumables 3 to 24 h required for completing the test	Initial equipment costs Can give false positive results for strains with intermediate resistance

Wayne P. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; 29th Ed. CLSI Supplement M100. Clin Lab Stand Institute; 2019.

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