MALDI-TOF MS for rapid detection and differentiation between Tet(X)-producers and non-Tet(X)-producing tetracycline-resistant Gram-negative bacteria

Figures & data

Table 1. Bacterial strain characteristics and corresponding the MALDI^Tet(X)-plus test results

			MIC						MS Ratio
Species	n	genes	TC(1s)^a	OTC(1s)^a	DOX(2s)^a	TGC(3s)^a	ERA(4s)^a	OMA(4s)^a	TGC	OTC
Control strains	4
E. coli-JM109-pBAD24-tet(X3)	1	tet(X3)	64	128	32	4	4	16	0.5 ± 0.09	0.2283 ± 0.0503
E. coli-JM109-pBAD24-tet(X4)	1	tet(X4)	64	128	16	8	2	16	0.43 ± 0.11	0.2155 ± 0.013
E. coli JM109 – pBAD24	1	-	2	2	0.5	0.03	0.008	0.125	0 ± 0	0.1346 ± 0.0629
E. coli 25,922	1	-	2	2	0.5	0.03	0.06	0.25	0 ± 0	0.1221 ± 0.0498
Test strains	90
Non-Tet(X)-producers in tetracycline-resistant strains	61
Acinetobacter spp.	2	tet(A)	>256	>256	128–256	2–4	1–2	8	0 ± 0	0.0041 ± 0.0058 to 0.0151 ± 0.0132
Acinetobacter spp.	6	tet(B)	>256	>256	128–256	0.5–4	0.5–1	0.5–8	0 ± 0	0 ± 0 to 0.0463 ± 0.0208
E. coli	17	tet(A)	64->256	64->256	4->256	0.06–4	0.03–2	0.25–8	0 ± 0	0 ± 0 to 0.0189 ± 0.0157
E. coli	7	tet(B)	128->256	128->256	32->256	0.125–2	0.03–1	0.5–8	0 ± 0	0 ± 0 to 0.0158 ± 0.0224
E. coli	1	tet(G)	256	256	64	2	0.5	8	0 ± 0	0 ± 0
E. coli	4	tet(M)	128–256	128->256	32–64	0.125–0.5	0.125–0.25	2	0 ± 0	0 ± 0
E. coli	4	tet(A)- tet(B)	128->256	>256	64–256	0.03–0.5	0.03–0.25	0.25–4	0 ± 0 to 0.0035 ± 0.005	0 ± 0 to 0.0111 ± 0.0157
E. coli	4	tet(A)- tet(M)	64–256	64->256	32->128	0.25–0.5	0.125–0.25	2–4	0 ± 0 to 0.0085 ± 0.0121	0 ± 0 to 0.0727 ± 0.0748
K. pneumoniae	3	tet(A)	128->256	256->256	32–256	0.5–4	0.125–2	4–8	0 ± 0 to 0.0033 ± 0.0047	0 ± 0 to 0.0518 ± 0.0309
K. pneumoniae	1	tet(D)	256	>256	128	2	1	8	0 ± 0	0.0231 ± 0.0326
K. pneumoniae	3	tet(A)- tet(D)	>256	>256	>256	1–2	1	8	0 ± 0	0.0128 ± 0.0137 to 0.0419 ± 0.0326
S. enteritis	5	tet(B)	64–256	128->256	8–64	0.5–2	0.06–0.5	1–4	0 ± 0	0 ± 0
S. enteritis	2	tet(A)- tet(B)	128	>256	64	1	0.25	2	0.0003 ± 0.0004 to 0.0003 ± 0.0005	0 ± 0 to 0.0083 ± 0.0117
S. enteritis	1	tet(B)- tet(D)	256	128	64	1	0.25	4	0 ± 0	0 ± 0
S. enteritis	1	tet(B)- tet(M)	256	>256	64	1	0.125	2	0 ± 0	0.0136 ± 0.0193
Tetracycline-susceptible strains	29
E. coli	29	non-tet(X)^b	0.5–1	1–2	1	0.125–0.5	0.03–0.06	0.25–1	0 ± 0 to 0.0018 ± 0.0025	0.0671 ± 0.0227 to 0.3897 ± 0.0184

TC, tetracycline; OTC oxytetracycline; DOX, doxycycline; TGC, tigecycline; ERA, eravacycline; OMA, omadacycline.

^aThe number in parentheses indicates the generation of tetracycline.

^bNon-tet(X) strains lack all tet genes as well as tet(X).

Table 2. Characteristics of test strains used for test validation

			MIC						MS Ratio
Species	n	genes	TC(1s)^a	OTC(1s)^a	DOX(2s)^a	TGC(3s)^a	ERA(4s)^a	OMA(4s)^a	TGC	OTC
Test strains	229
Tet(X)-producers	124
Acinetobacter spp.	38	tet(X3)	64->256	16->256	1–128	16–64	4–32	4–64	0.0134 ± 0.0038 to 0.5778 ± 0.1437	0.0316 ± 0.0089 to 0.2793 ± 0.1123
Acinetobacter spp.	1	tet(X2)-tet(X6)	>256	256	128	32	4	16	0.231 ± 0.1276	0.2656 ± 0.0449
Acinetobacter spp.	16	tet(X3)-tet(X6)	32->256	8->256	8–128	16–64	4–16	4–64	0.0117 ± 0.0165 to 0.3845 ± 0.1291	0.0415 ± 0.0257 to 0.1987 ± 0.0493
E. coli	69	tet(X4)	32->256	32->256	32–128	1–32	1–16	8–64	0.0008 ± 0.0011 to 0.4812 ± 0.0918	0.0104 ± 0.0089 to 0.2097 ± 0.0298
Non-Tet(X)-producing tetracycline-resistant strains	88
Acinetobacter spp.	6	tet(B)	>256	>256	64–256	2–4	0.25–1	2–8	0 ± 0	0 ± 0 to 0.0271 ± 0.0192
Acinetobacter spp.	1	tet(M)	>256	>256	128	2	1	4	0 ± 0	0.0094 ± 0.0133
Acinetobacter spp.	1	tet(A)- tet(B)-tet(D)	>256	>256	64	2	1	4	0 ± 0	0 ± 0
E. coli	32	tet(A)	64–256	64->256	16–256	0.125–2	0.06–2	0.125–8	0 ± 0	0 ± 0 to 0.0344 ± 0.0255
E. coli	9	tet(B)	256->256	256->256	64->256	0.25–1	0.06–0.5	0.25–8	0 ± 0	0 ± 0 to 0.0704 ± 0.0629
E. coli	3	tet(D)	256	64->256	32–128	0.25–4	0.25–2	4–8	0 ± 0	0 ± 0
E. coli	1	tet(M)	128	256	128	0.25	0.125	2	0 ± 0	0 ± 0
E. coli	1	tet(A)- tet(B)	64	64	16	0.125	0.25	2	0 ± 0	0 ± 0
E. coli	9	tet(A)- tet(M)	256->256	64->256	32->256	0.25–2	0.06–1	1–4	0 ± 0 to 0.0147 ± 0.0208	0 ± 0 to 0.0287 ± 0.0113
E. coli	2	tet(B)- tet(D)	256->256	>256	32->256	0.25–4	0.25–2	4–16	0 ± 0 to 0.0005 ± 0.0008	0 ± 0 to 0.0237 ± 0.0241
E. coli	1	tet(B)- tet(M)	>256	>256	256	0.5	0.125	1	0 ± 0	0.0235 ± 0.0332
E. coli	6	tet(D)- tet(M)	128->256	>256	32->256	0.25–4	0.25–3	2–8	0 ± 0	0 ± 0 to 0.0939 ± 0.0332
E. coli	1	TMexCD1- TOprJ1	16	16	32	4	8	16	0 ± 0	0.0581 ± 0.0170
K. pneumoniae	6	tet(A)	256->256	>256	64	0.25–0.5	0. 25–0.5	2–4	0 ± 0 to 0.0168 ± 0.0237	0 ± 0 to 0.017 ± 0.024
K. pneumoniae	1	tet(B)	128	256	256	0.5	0.125	1	0 ± 0	0 ± 0
K. pneumoniae	1	tet(A)- tet(M)	128	128	32	0.5	0.25	2	0 ± 0	0 ± 0
S. enteritis	7	tet(B)	128	128->256	32–64	0.5	0.06–0.125	2–4	0 ± 0	0 ± 0 to 0.006 ± 0.0085
Tetracycline-susceptible strains	17
E. coli	10	non-tet(X)^b	0.5–1	2	1	0.125–0.25	0.03–0.06	0.5	0 ± 0 to 0.0004 ± 0.0005	0.0465 ± 0.0103 to 0.3929 ± 0.1155
K. pneumoniae	7	non-tet(X)^b	4–8	4–8	2–4	0.5–1	0.25–0.5	1–4	0 ± 0	0.0619 ± 0.0104 to 0.2202 ± 0.0255

TC, tetracycline; OTC oxytetracycline; DOX, doxycycline; TGC, tigecycline; ERA, eravacycline; OMA, omadacycline.

^aThe number in parentheses indicates the generation of tetracycline.

^bNon-tet(X) strains lack all tet genes as well as tet(X).

Figure 1. Strategy for identification of Tet(x)-producers and non-Tet(X)-producing tetracycline-resistant strains and tetracycline-susceptible strains using the MALDI^Tet(X)-plus test. Strains that showed peaks of tigecycline metabolite or oxytetracycline metabolite were labeled with “+”

Figure 2. Representative MALDI-TOF MS results for detection of Tet(x)-producers and non-Tet(X)-producers. (a) Structures of TGC and OTC along with their oxygen-modified derivatives. (b) Representative MALDI-TOF MS spectra of tigecycline and oxytetracycline oxygenation assays after a 3 h incubation. Peaks of tigecycline and tigecycline metabolite are denoted by dashed red lines and represent peaks at m/z 586 ± 0.2 and m/z 602 ± 0.2, respectively. Peaks of oxytetracycline and oxytetracycline metabolite are denoted by dashed blue lines and represent peaks at m/z 460 ± 0.2 and m/z 476 ± 0.2, respectively

Figure 3. MALDI^Tet(x)-plus test results using 319 test strains. (a) The cutoff value of 0.00405 can clearly distinguish Tet(X)-producers M/(M + T) >0.00405 (b) The cutoff value of 0.05945 can clearly identify non-Tet(X)-producers possessing M/(M + O) <0.05945. Three independent experiments were performed

Figure 4. LC-MS/MS results for degradation of OTC by Tet(x)-producers, non-Tet(X) producers in tetracycline-resistant strains, and tetracycline-susceptible strains. The control group includes only OTC. We compare tetracycline-susceptible strains with Tet(X)-producers, non-Tet(X) producers in tetracycline-resistant strains, and the control group (three asterisk for p < 0.001)

Figure5 Strain survival from 50 mg/L OTC, after pre-treated H₂O₂ for 30 min and compared with non-pre-treated strain (one asterisk for p < 0.05). control group grown in LB medium without H₂O₂ and OTC. Curves represent the mean of five independent cultures per time point

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.