Cefmetazole Resistance Mechanism for Escherichia Coli Including ESBL-Producing Strains

Figures & data

Table 1 Primers Used in This Study

Primer	Sequence (5’→3’)	Reference
ompF-F	AAGTAGTAGGTTGCGCCCAC	[10]
ompF-R	AGTTCGATTTCGGTCTGCGT
ompC-F	ATTCTGGCAGTACGTCGGTC	[10]
ompC-R	AAACAACTCCTGGACCCGTG
phoE-F	CAGGCTTCCACGTCATACAA	This study
phoE-R	TTATGGTCGTTGGGAAGCTG
ampC-F	TCAAACCAGACGGCTTCACA	[11]
ampC-R	GTCTGTATGCCAACTCCAGTATCG
acrA-F	CTTAGCCCTAACAGGATGTG	[12]
acrA-R	TCTTTGAAATTACGCTTCAGG
yhiV-F	CCGTACCGGTGGTTATTCTC	[12]
yhiV-R	ATCGATTTATGCGTCGCTTC
mdfA-F	CATTGGCAGCGATCTCCTTT	[12]
mdfA-R	TATAGTCACGACCGAGTTCTTTC
gapA-F	GGCCAGGACATCGTTTCCAA	[11]
gapA-R	TCGATGATGCCGAAGTTATCGTT

Figure 1 Resistance to in vitro cefmetazole exposure, by MIC group, in E. coli total (A), ESBL-producing E. coli (B), and non-ESBL E. coli (C). The data shows the cumulative resistance rate and 95% CI due to everyday cefmetazole exposure.

Note: *P < 0.05.

Table 2 MIC of Cefmetazole Against Escherichia Coli

Strain Number	MIC (μg/mL) of Cefmetazole
	Wild Type	1st Subculture 10 Days	Cefmetazole Free for 14 Days	2nd Subculture 10 Days
<ESBL>
ES-1	1	128	64	512
ES-2	1	4	2	128
ES-3	1	64	2	64
ES-4	1	64	64	256
ES-5	1	4	2	128
ES-6	1	64	16	128
ES-7	2	64	64	256
ES-8	2	64	8	256
ES-9	2	128	16	512
ES-10	2	8	2	32
ES-11	2	64	64	64
ES-12	4	32	2	32
ES-13	4	64	16	64
ES-14	4	256	16	256
<non-ESBL>
EC-1	0.5	8	2	64
EC-2	1	4	4	32
EC-3	1	2	1	16
EC-4	1	4	1	4
EC-5	1	64	2	128
EC-6	1	4	0.5	64
EC-7	2	8	2	128
EC-8	2	64	16	128
EC-9	2	128	8	128
EC-10	4	64	16	128
EC-11	4	64	16	128
ATCC25922	1	32	4	128

Figure 2 mRNA expression levels of ompF, ompC, and phoE in resistant and non-resistant strains after 10 days of cefmetazole exposure.

Note: *P < 0.05.

Abbreviations: R, cefmetazole resistant group; non-R, cefmetazole non-resistant group.

Figure 3 mRNA expression levels of chromosomal ampC, acrA, yhiV, mdfA genes in this study cefmetazole exposure assay.

Notes: The expression levels of ampC, acrA, yhiV, mdfA were expressed with each wild type as 1.

Abbreviations: R, cefmetazole resistant group; non-R, cefmetazole non-resistant group.

Figure 4 Resistance to in vitro cefmetazole exposure at the time of addition of relebactam.

Note: *P < 0.05.

Abbreviations: CMZ, cefmetazole; REL, relebactam.

Table 3 Changes in ompF, ompC and phoE mRNA Expression in Combination with Relebactam and Cefmetazole

Strain Number	Wild Type	CMZ Subculture 10 Days				CMZ With REL (16 µg/mL) Subculture 5 Days
	MIC (μg/mL)	MIC (μg/mL)	mRNA Expression Level (fold)			MIC (μg/mL)	mRNA Expression Level (fold)
			ompF	ompC	phoE		ompF	ompC	phoE
<CMZ resistance>
ES-4	1	64	0.78	0.49	0.37	4	3.2	8	1.8
ES-8	2	64	0.11	0.23	0.31	2	2.1	1.52	0.3
ES-14	4	256	0.2	0.12	0.49	4	3.3	2.7	1.9
EC-9	2	128	0.08	0.2	0.007	2	4.37	1.6	5.9
EC-10	4	64	0.6	0.9	0.6	4	3.46	0.65	0.2
<non-resistance>
EC-1	0.5	8	1	0.2	0.1	4	1.7	0.8	0.2
EC-4	1	4	1.7	0.8	0.2	4	5.2	1.5	1
EC-7	2	8	7.5	0.7	0.4	2	2.7	1.36	0.3
ES-5	1	4	2.17	0.01	0.54	8	1.4	1.6	0.2
ES-12	4	32	1.1	1.19	0.05	4	2.3	1.1	0.2

Notes: The expression levels of ompF, ompC, and phoE were expressed with each wild type as 1.

Abbreviations: CMZ, cefmetazole; REL, relebactam; EC, ESBL-non-producing E. coli; ES, ESBL-producing E. coli.

Chong Y, Shimoda S, Yakushiji H, et al. Community spread of extended-spectrum β-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis: a long-term study in Japan. J Med Microbiol. 2013;62(Pt7):1038–1043. PMID: 23538565. doi:10.1099/jmm.0.059279-0

 PubMed Web of Science ®Google Scholar

Doi Y, Park YS, Rivera JI, et al. Community-associated extended-spectrum β-lactamase-producing Escherichia coli infection in the United States. Clin Infect Dis. 2013;56(5):641–648. PMID: 23150211; PMCID: PMC3563390. doi:10.1093/cid/cis942

 PubMed Web of Science ®Google Scholar

Tamma PD, Aitken SL, Bonomo RA, Mathers AJ, van Duin D, Clancy CJ. Infectious Diseases Society of America guidance on the treatment of extended-spectrum β-lactamase producing Enterobacterales (ESBL-E), carbapenem-resistant Enterobacterales (CRE), and Pseudomonas aeruginosa with difficult-to-treat resistance (DTR-P. aeruginosa). Clin Infect Dis. 2021;72(7):e169–e183. PMID: 33106864. doi:10.1093/cid/ciaa1478

 PubMed Web of Science ®Google Scholar