Molecular Epidemiology and Mechanisms of High-Level Resistance to Meropenem and Imipenem in Pseudomonas aeruginosa

Figures & data

Table 1 Primers Used in This Study

Primer	Sequence (5ʹ-3ʹ)	Size (bp)	References
blaIMP1	F: CTACCGCAGCAGAGTCTTTG	587	[^Citation17]
	R: AACCAGTTTTGCCTTACCAT
blaIMP2	F: GTTTTATGTGTATGCTTCC	678	[^Citation18]
	R: AGCCTGTTCCCATGTAC
blaVIM1	F:AGTGGTGAGTATCCGACAG	261	[^Citation19]
	R:ATGAAAGTGCGTGGAGAC
blaVIM2	F: ATGTTCAAACTTTTGAGTAAG	801	[^Citation20]
	R: CTACTCAACGACTGAGCG
blaNDM	F: GGTTTGGCGATCTGGTTTTC	621	[^Citation21]
	R: CGGAATGGCTCATCACGATC
blaSPM	F: CCTACAATCTAACGGCGACC	650	[^Citation22]
	R: TCGCCGTGTCCAGGTATAAC
oprD	F:CTACGGCTACGGCGAGGAT	NA	[^Citation23]
	R:GACCGGACTGGACCACGTACT
mexA	F:AACCCGAACAACGAGCTG
	R:ATGGCCTTCTGCTTGACG
mexC	F:GGAAGAGCGACAGGAGGC
	R:CTGCACCGTCAGGCCCTC
mexE	F:TACTGGTCCTGAGCGCCT
	R:CAGCGGTTGTTCGATGA
mexY	F:CCGCTACAACGGCTATCCCT		[^Citation24]
	R:AGCGGGATCGACCAGCTTTC
rpsL	F:GCAACTATCAACCGACTGGTG		[^Citation25]
	R:GCTGTGCTCTTGCAGGTTGTG

Table 2 MIC and Characterization of Efflux Pump Overexpression and Carbapenemase Genes in the Carbapenem-Resistant P.aeruginosa Isolates

ID	Meropenem MIC (µg/mL)	Imipenem MIC (µg/mL)	mexA	mexC	mexE	mexY	OprD	Genotypic Profile (Different MBLs Genes)
PA- 1	2	16	-	-	-	-	-	blaVIM1
PA- 2	256	256	-	+	-	-	-	blaVIM1
PA- 3	256	256	-	+	-	+	-	blaVIM1
PA- 4	256	256	-	-	-	+	-	blaVIM1
PA- 5	256	256	+	+	-	+	-	blaVIM1- blaNDM
PA- 6	2	8	-	+	-	+	-	blaVIM1
PA- 7	256	256	-	+	-	+	-	blaVIM1
PA- 8	512	512	-	+	-	-	-	–
PA- 9	256	256	+	+	+	+	-	blaVIM1
PA- 10	256	256	-	+	-	-	-	blaVIM1
PA- 11	256	256	-	+	-	+	-	blaVIM1
PA- 12	512	512	-	+	-	+	-	blaVIM1
PA- 13	256	256	-	+	-	+	-	blaVIM1
PA- 14	256	256	-	-	+	-	-	blaVIM1
PA- 15	2	16	+	+	-	-	-	blaVIM1
PA- 16	512	512	-	+	+	-	-	blaVIM1- blaNDM
PA- 17	256	256	-	+	-	-	-	blaVIM1- blaNDM
PA- 18	256	256	-	-	-	-	-	blaVIM1
PA- 19	512	512	+	+	+	+	-	blaVIM1
PA- 20	512	512	+	+	-	+	-	blaVIM1
PA- 21	256	256	-	-	+	+	-	blaVIM1
PA- 22	256	128	+	+	-	+	-	blaVIM1
PA- 23	256	256	-	+	-	+	-	blaVIM1
PA- 24	8	2	-	-	-	+	-	blaVIM1
PA- 25	512	512	-	+	-	+	-	blaVIM1
PA- 26	256	256	-	+	-	+	-	blaVIM1
PA- 27	256	256	-	+	-	+	-	blaVIM1
PA- 28	256	256	-	+	-	-	-	blaVIM1
PA- 29	256	256	-	+	-	-	-	blaVIM1-blaNDM
PA- 30	256	256	+	+	+	+	-	blaVIM1
PA- 31	256	256	-	+	-	+	-	blaVIM1
PA- 32	256	256	-	-	-	-	-	blaVIM1
Total No.			7	25	6	20	0
Total percent (%)*	21.8	78	18.7	62.5	0

Notes: *Percentages were calculated to total number of carbapenem-resistant P.aeruginosa isolates.

Abbreviations: ID, identification number; PA, Pseudomonas aeruginosa; MIC, minimum inhibitory concentration; ABM, MexAB-OprM; CDJ, MexCD-OprJ; EFN, MexEF-OprN; XY, MexXY-OprM; OprD, Outer membrane porin; -, absent; +, present; IMP1, bla_IMP1; IMP2, bla_IMP2; VIM1, bla_VIM1; VIM2, bla_VIM2; NDM, bla_NDM; SPM, bla_SPM.

Table 3 Correlation of Efflux Pump Gene Expression with Carbapenem-Resistant and Sensitive P.aeruginosa Isolates

Efflux Pump Gene Expression		Meropenem					Imipenem
		Sensitive (No. = 35)		Resistant (No. = 29)		P value	Sensitive (No. = 32)		Resistant (No. = 32)		P value
		No.	%*	No.	%*		No.	%*	No.	%*
mexY	Not over- expressed	30	85.7	11	37.9	<0.001	28	87.5	13	40.6	<0.001
Over- expressed	5	14.3	18	62.1	4	12.5	19	59.4
mexA	Not over- expressed	25	71.4	23	79.3	0.469	23	71.9	25	78.1	0.564
Over- expressed	10	28.6	6	20.7	9	28.1	7	21.9
mexC	Not over- expressed	27	77.1	6	20.7	<0.001	0 26	0 81.2	7	21.9	<0.001
Over- expressed	8	22.9	23	79.3	6	18.8	25	78.1
mexE	Not over- expressed	29	82.9	23	79.3	0.717	0 26	0 81.2	26	81.2	1.000
Over- expressed	6	17.1	6	20.7	6	18.8	6	18.8

Notes: *Percentages were correlated to total No. of either carbapenem-resistant or sensitive P.aeruginosa isolates.

Table 4 Bivariate Correlation Analysis Between Meropenem, Imipenem Resistance and Efflux Pump Over-Expression

Efflux Pump		Meropenem	Imipenem
XY	(r)	0.363	0.327
P	0.003	0.008
ABM	(r)	−0.114-	−0.141-
P	0.370	0.267
CDJ	(r)	0.532	0.564
P	<0.001	<0.001
EFN	(r)	0.009	−0.031-
P	0.947	0.805

Note: Significant correlation at P value <0.05.

Abbreviation: r, Spearman’s rho correlation.

Figure 1 Dendrogram based on RAPD analysis of 32 P.aeruginosa isolates. The dendrogram was developed using gelJ software.

Abbreviation: PA, Pseudomonas aeruginos.

Senda K, Arakawa Y, Ichiyama S, et al. PCR detection of metallo-beta-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum beta-lactams. J Clin Microbiol. 1996;34(12):2909–2913. doi:10.1128/JCM.34.12.2909-2913.19968940421

 PubMed Web of Science ®Google Scholar

Shibata N, Doi Y, Yamane K, et al. PCR typing of genetic determinants for metallo-beta-lactamases and integrases carried by gram-negative bacteria isolated in Japan, with focus on the class 3 integron. J Clin Microbiol. 2003;41(12):5407–5413. doi:10.1128/JCM.41.12.5407-5413.200314662918

 PubMed Web of Science ®Google Scholar

Tsakris A, Pournaras S, Woodford N, et al. Outbreak of infections caused by Pseudomonas aeruginosa producing VIM-1 carbapenemase in Greece. J Clin Microbiol. 2000;38(3):1290–1292. doi:10.1128/JCM.38.3.1290-1292.200010699045

 PubMed Web of Science ®Google Scholar

Poirel L, Naas T, Nicolas D, et al. Characterization of VIM-2, a carbapenem-hydrolyzing metallo-beta-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob Agents Chemother. 2000;44(4):891–897. doi:10.1128/AAC.44.4.891-897.200010722487

 PubMed Web of Science ®Google Scholar

Nordmann P, Poirel L, Carrer A, Toleman MA, Walsh TR. How to detect NDM-1 producers. J Clin Microbiol. 2011;49(2):718–721. doi:10.1128/JCM.01773-1021123531

 PubMed Web of Science ®Google Scholar

Queenan AM, Bush K. Carbapenemases: the versatile beta-lactamases. Clin Microbiol Rev. 2007;20(3):440–458. doi:10.1128/CMR.00001-0717630334

 PubMed Web of Science ®Google Scholar

Quale J, Bratu S, Gupta J, Landman D. Interplay of efflux system, ampC, and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother. 2006;50(5):1633–1641. doi:10.1128/AAC.50.5.1633-1641.200616641429

 PubMed Web of Science ®Google Scholar

Yoneda K, Chikumi H, Murata T, et al. Measurement of Pseudomonas aeruginosa multidrug efflux pumps by quantitative real-time polymerase chain reaction. FEMS Microbiol Lett. 2005;243(1):125–131. doi:10.1016/j.femsle.2004.11.04815668010

 PubMed Web of Science ®Google Scholar

Shigemura K, Osawa K, Kato A, et al. Association of overexpression of efflux pump genes with antibiotic resistance in Pseudomonas aeruginosa strains clinically isolated from urinary tract infection patients. J Antibiot (Tokyo). 2015;68(9):568–572. doi:10.1038/ja.2015.3425850341

 PubMed Web of Science ®Google Scholar