Efflux Pump AcrAB Confers Decreased Susceptibility to Piperacillin–Tazobactam and Ceftolozane–Tazobactam in Tigecycline-Non-Susceptible Klebsiella pneumoniae

Figures & data

Table 1 Primers Used in This Study

Primers	Sequence (5′ to 3′)	Product (bp)	Reference
GmF	CGAATTAGCTTCAAAAGCGCTCTGA	1649	^Citation24
Gm-R2	AATTGGGGATCTTGAAGTTCCT
EBGNHe-5	CCCGCTAGCGAAAAGATGTTTCGTGAAGC	1960	^Citation24
EBGh3-3	GGGAAGCTTATTATCGTGAGGATGCGTCA
acrB-UF-F	CGGCAAAGCGAAAGTGGAG	512	This study
acrB-UF-R	TCAGAGCGCTTTTGAAGCTAATTCG^a GAAATTAGGCATGTCTTAACGGC
acrB-DF-F	AGGAACTTCAAGATCCCCAATT^b GAGCATCATTAATCTTCACTCC	509	This study
acrB-DF-R	GTA TTC GTC CAT AAC GCA TC
acrB-internal-F	TATCGGCTACGACTGGACC	317	This study
acrB-internal-R	GCCCTTTGCCCTCTTTCT
acrB-external-F	TGATTTCCTGCGCCTGAAG	4247	This study
acrB-external-R	AGCGTTTCG CAG AGAAAG C
ramR-romA-F	GATGGCGACCACGCTGAA	305	This study
ramR-romA-R	TATGCCGACTGGGCGAAA
Hindш-acrB-F	ATGACCATGATTACGCCAAGCTTATGCCTAATTTCTTTATCGATCG	3192	This study
BamHⅠ-acrB-R	GTACCCCATCGATGGGGGATCCTTAATGATGCTCAACCTGATGGC

Notes: ^aReverse-complement to Gm-F; ^breverse-complement to Gm-R; Gm-F/R for hygromycin B resistance gene hph cassette flanked by the Flp recombinase target sites; EBGNHe-5/EBGh3-3 for the recombination vector pKOBEG; acrB-UF-F/R for 5ʹ upstream flanking of acrB fragment; acrB-DF-F/R for 3ʹ downstream flanking of acrB fragment; acrB-internal-F/R for a fragment inside acrB; acrB-external-F/R for a fragment including UF, DF, and acrB.

Table 2 Antimicrobial Susceptibility Patterns of the K. pneumoniae Clinical Parental Strains and Their Derivatives

Strains								MIC (μg/mL)
	PIP	TZP	C/T	SAM	CFZ	CXM	CAZ	CCV	CZA	SCF	FEP	MEM	IPM	ATM	AMK	TGC	CIP	CST
TNSKP24^a	256	4/4	1/4	32/16	>32	>64	0.5	0.5/0.25	0.25/4	16/8	8	≤0.06	0.125	2	0.5	4	0.5	0.5
TNSKP24ΔacrB^b	256	0.25/4	0.25/4	32/16	>32	>64	0.5	0.5/0.25	0.25/4	16/8	8	≤0.06	0.125	2	0.5	0.5	≤0.06	0.5
TNSKP24ΔacrB/pHSG396	256	0.25/4	0.25/4	32/16	>32	>64	0.5	0.5/0.25	0.25/4	16/8	8	≤0.06	0.125	2	0.5	0.5	≤0.06	0.5
TNSKP24ΔacrB/pHSG396acrB	256	2/4	0.5/4	32/16	>32	>64	0.5	0.5/0.25	0.25/4	16/8	8	≤0.06	0.125	2	0.5	2	0.5	0.5
K2606^c	512	2/4	0.25/4	32/16	>32	>64	1	0.5/0.25	0.25/4	16/8	4	≤0.06	≤0.06	4	2	2	1	1
K2606-4^d	512	16/4	1/4	32/16	>32	>64	2	0.5/0.25	0.25/4	32/16	8	≤0.06	≤0.06	8	2	>128	8	1
K2606-4ΔacrB^e	256	2/4	0.25/4	32/16	>32	>64	2	0.5/0.25	0.25/4	32/16	8	≤0.06	≤0.06	8	2	2	≤0.06	1
K2606-4ΔacrB/pHSG396	256	2/4	0.25/4	32/16	>32	>64	2	0.5/0.25	0.25/4	32/16	8	≤0.06	≤0.06	8	2	2	≤0.06	1
K2606-4ΔacrB/pHSG396acrB	512	8/4	0.5/4	32/16	>32	>64	2	0.5/0.25	0.25/4	32/16	8	≤0.06	≤0.06	8	2	16	16	1
K2606-16^f	512	16/4	1/4	32/16	>32	>64	2	0.5/0.25	0.25/4	32/16	8	≤0.06	≤0.06	8	2	>128	8	1
K2606-16ΔacrB^g	256	2/4	0.25/4	32/16	>32	>64	2	0.5/0.25	0.25/4	32/16	8	≤0.06	≤0.06	8	2	2	≤0.06	1
K2606-16ΔacrB/pHSG396	256	2/4	0.25/4	32/16	>32	>64	2	0.5/0.25	0.25/4	32/16	8	≤0.06	≤0.06	8	2	2	≤0.06	1
K2606-16ΔacrB/pHSG396acrB	512	8/4	0.5/4	32/16	>32	>64	2	0.5/0.25	0.25/4	32/16	8	≤0.06	≤0.06	8	2	16	16	1
ATCC25922	2	2/4	0.125/4	4/2	4	4	0.125	0.25/0.125	≤0.25/4	0.25/0.125	≤0.06	≤0.06	≤0.06	≤0.06	2	0.125	≤0.06	0.5

Notes: ^aTNSKP24, a clinical K. pneumoniae strain with tigecycline MIC 4 μg/mL. ^bTNSKP24ΔacrB, TNSKP24 without acrB. ^cK2606, a clinical K. pneumoniae strain with tigecycline MIC 2 μg/mL. ^dK2606-4, K2606 exposure to tigecycline with a concentration 4× MIC of K2606. ^eK2606-4ΔacrB, K2606-4 without acrB. ^fK2606-16, K2606 exposure to tigecycline with a concentration 16× MIC of K2606. ^gK2606-16ΔacrB, K2606-16 without acrB.

Abbreviations: PIP, piperacillin; TZP, piperacillin–tazobactam; C/T, ceftolozane–tazobactam; SAM, ampicillin-sulbactam; CFZ, cefazolin; CXM, cefuroxime; CAZ, ceftazidime; SCF, cefoperazone-sulbactam; CCV, ceftazidime-clavulanate; CZA, ceftazidime-avibactam; FEP, cefepime; MEM, meropenem; IPM, imipenem; ATM, aztreonam; AMK, amikacin; TGC, tigecycline; CIP, ciprofloxacin; CST, colistin.

Table 3 Genomic Changes of K2606 After Exposure to Tigecycline

Location	Mutation	Position	Mutation Type	Information	Annotation	Gene Description
SNP^a in both K2606-4 and K2606-16
Chr^b	T843830G	Intergenic of gene0838 and gene0839	-	-	fimB;-	type 1 fimbriae regulatory protein; hypothetical protein
Chr	A1127404G, A1127631G A1127769G, A1128074G	Intergenic of gene1117 and gene1123	-	-	csrA;-	carbon storage regulator; phospholipid-binding domain protein
Chr	C3872385 T	Intergenic of gene3810 and gene3811	-	-	romA; ramR	beta-lactamase domain protein; TetR family transcriptional regulator
pA^c	A90269C	Internal of gene0095	Nonsynonymous	c.T751G p.S251A	tetA	tetracycline efflux MFS transporter
SNP in K2606-4 only
Chr	G303519A	Intergenic of gene0279 and gene0278	-	-	tusA;-	tRNA 2-thiouridine (34) synthase; hypothetical protein
Chr	G586814C	Intergenic of gene0574 and gene0575	-		-;-	hypothetical protein; hypothetical protein
Chr	G4324175T	Internal of gene4263	Nonsynonymous	c.G386T, p.R129L	dksA	transcriptional regulator
Chr	C4350054A	Internal of gene4291	Nonsynonymous	c.G59T, p.C20F	-	hypothetical protein
pB^d	C125391G	Intergenic of gene0112 and gene0113	-		-;higA	hypothetical protein; transcriptional regulator
SNP in K2606-16 only
Chr	C427705A	gene0395	Nonsynonymous	c. C116A, p.P39Q	rpsJ	ribosome protein RpsJ
Chr	C2049592G	Intergenic of gene1998 and gene1999	-	-	-;-	GNAT family acetyltransferase; transcriptional regulator
Chr	A4052373C	Internal of gene3986	Nonsynonymous	c. A101C, p.Y34S,	-	hypothetical protein
pD^e	C29141G	Intergenic of gene0036 and gene0037	-	-	-;-	hypothetical protein; hypothetical protein
^fINDEL in K2606-4
Chr	GCTATACCAAA deleted between 586,798 and 586,808	Intergenic of gene0572 and gene0573	Deletion	-	glxK;-	glycerate 2-kinase; hypothetical protein

Notes: ^aSNP, single nucleotide polymorphism; ^bChr, chromosome; ^cpA, plasmid A; ^dpB, plasmid B; ^epD, plasmid D; ^fINDEL, insertion-deletion.

Figure 1 Gene mutation and expression in TNSKP24, K2606 K2606-4, and K2606-16. (A) Mutations in ramR and the intergenic region of ramR-romA in K. pneumoniaeK2606 and its two derivatives when compared with K. pneumoniae MGH78578 (GenBank: CP000647.1). The arrow segments in the intergenic region of romR-romA are the inverted repeat (IR) sequences recognized by the RamR protein (24). The numbering system is based on the “t” prior to romA’s start codon ATG as the 194. Missense mutation (D77H) in ramR, and point mutation (C111T) in the intergenic region of ramR-romA was detected in K2606, K2606-4, and K2606-16, and an additional novel point mutation (C133T) was found between the palindromic repeats in the 2 derivatives. (B) and (C) Transcriptional expression (mean±standard deviation) of ramA, acrB, rarA, and oqxB in TNSKP24, K2606 K2606-4, and K2606-16.

Bi D, Jiang X, Sheng Z-K, et al. Mapping the resistance-associated mobilome of a carbapenem-resistant Klebsiella pneumoniae strain reveals insights into factors shaping these regions and facilitates generation of a ‘resistance-disarmed’ model organism. J Antimicrob Chemother. 2015;70(10):2770–2774. doi:10.1093/jac/dkv20426169555

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