Characterization of Two Macrolide Resistance-Related Genes in Multidrug-Resistant Pseudomonas aeruginosa Isolates

Gene	Primer	Sequence (5’→3’)	Purpose	Restriction endonuclease	Vector	Amplicon size (bp)	Annealing temperature
mphE	mphE-SF	ATGCCCAGCATATAAATCGC	Screening			271	60°C
	mphE-SR	ATATGGACAAAGATAGCCCG
	mphE-OF	CGGAATTCTATTCAAAAAAACTTATCCGACTTA	Cloning	EcoR I	pUCP18	885	60°C
	mphE-OR	CCAAGCTTTTATATAACTCCCAACTGAGCTTTT		Hind III
msrE	msrE-SF	TATAGCGACTTTAGCGCCAA	Screening			395	62°C
	msrE-SR	GCCGTAGAATATGAGCTGAT
	msrE-OF	CGGAATTCTTTTTGGGAGGACACTGTGATGCTA	Cloning	EcoR I	pUCP18	1,467	62°C
	msrE-OR	CCAAGCTTTTATATAACTCCCAACTGAGCTTTT		Hind III

Table II

The MIC values for 13 antibiotics against 262 clinical Pseudomonas aeruginosa isolates.

Antibiotics	MIC range (μg/ml)	MIC50 (μg/ml)	MIC90 (μg/ml)	Resistance (%)
Cefuroxim	0.5–32	16	> 32	44.1
Cefepime	1–64	8	64	36.7
Meropenam	0.0125–32	4	32	30.1
Ceftazidime	1–64	16	> 64	52.1
Gentamicin	0.125–64	16	64	32.1
Tobramycin	0.5–32	4	> 32	29.6
Amikacin	>256	2	> 256	34.9
Netilmicin	0.5–512	8	> 512	42.1
Colistin	0.25–16	2	> 16	28.2
Azithromycin	0.5–256	64	> 256	78.1
Clarithromycin	1–1024	256	> 1024	69.5
Roxithromycin	0.5–1024	256	> 1024	76.8
Erythromycin	1–1024	512	> 1024	82.1

Table III

Coverage and abundance of the macrolide resistance genes in the pooled DNA from 262 Pseudomonas aeruginosa isolates.

Genotype	Reference sequence	Coverage^{^a}	Abundance^{^b}
mphE	AY522431	1.00	26.0
msrE	AY522431	1.00	24.0

1a – the ratio of the number of bases that mapped to the number of bases in the reference sequence

2b – the number of copies of the reference sequence in the genomic data

Table IV

MIC results for the recombinants, clinical strains, and controls (μg/ml).

Strain	ERY	KIT	ROX	CLR	AZM
pUCP18-msrE/DH5α (PAO2276)	512	256	1024	256	32
pUCP18-msrE/DH5α (PAO2883)	512	512	1024	256	32
pUCP18-mphE/DH5α (PAO1609)	512	256	1024	256	32
pUCP18-mphE/DH5α (PAO2883)	512	512	1024	256	32
PAO2883	1024	512	1024	512	> 128
PAO2276	512	256	1024	512	> 128
PAO1609	> 1024	256	1024	512	> 128
pUCP18/DH5α	128	256	128	32	4
DH5α	128	512	128	32	4
ATCC 27853	32	16	64	16	< 1

1 ERY – erythromycin, KIT – kitasamycin, ROX – roxithromycin, CLR – clarithromycin, AZM – azithromycin

Table V

Amino acid polymorphisms in the MsrE variants.

Accession No.	Amino acid position^{^a}							Reference
Accession No.	45	79	80	128	183	198	444	Reference
MG585957.1	Ser	Glu	Thr	Ser	Glu	Glu	Ile	Gonzalez-Plaza et al. 2018
MG585949.1	Ile				Asp			Gonzalez-Plaza et al. 2018
CP032136.1					Asp
LS992184.1		Gly			Asp
CP026233.1					Asp		Val	Weingarten et al. 2018
CP021960.1			Lys		Asp
CP011374.1				Gly	Asp	Lys
MsrE-2276					Asp			this study
MsrE-2883					Asp			this study

1a – amino acid positions using the protein sequence MG585957.1 as the reference

Table VI

Amino acid polymorphisms in the MphE variants.

Accession No.	Amino acid position^{^a}				Reference
Accession No.	17	28	133	231	Reference
CP029638.1	Ile	Ile	Glu	Thr	Beker et al. 2018
CP035931.1	Leu
KX443408.1	Leu		Asp
CP011374.1	Leu	Leu		Ile
mphE-1609	Leu				this study
mphE-2883	Leu				this study

1a – amino acid positions using the protein sequence CP029638.1 as the reference

Articles in this issue

DOI: https://doi.org/10.33073/pjm-2020-038 | Journal eISSN: 2544-4646 | Journal ISSN: 1733-1331

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Language: English

Page range: 349 - 356

Submitted on: May 14, 2020

Accepted on: Aug 15, 2020

Published on: Sep 8, 2020

Published by: Polish Society of Microbiologists

In partnership with: Paradigm Publishing Services

Publication frequency: 4 issues per year

Keywords:

Pseudomonas aeruginosa,

mphE,

Related subjects:

Microbiology and virology

© 2020 QING CHEN, WEI LU, DANYING ZHOU, GUOTONG ZHENG, HONGMAO LIU, CHANGRUI QIAN, WANGXIAO ZHOU, JUNWAN LU, LIYAN NI, QIYU BAO, AIFANG LI, TENG XU, HAILI XU, published by Polish Society of Microbiologists
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Volume 69 (2020): Issue 3 (September 2020)