Emergence of colistin-resistant hypervirulent Klebsiella pneumoniae (CoR-HvKp) in China

Figures & data

Table 1. Antimicrobial susceptibility profiles, isolates information and biofilm-production ability of colistin-resistance K. pneumoniae.

Name	Province	Gender	Age	Specimen type	MLST	Serotype	Hypervirluence factors	COL	AMK	FEP	FOX	CAZ	CRO	CIP	ETP	TGC	SXT	Biofilm formation (OD590nm)
A155	Hunan	M	59	bile	ST5253	K28	iroB iucA	4	≤8	≤1	≤4	≤1	≤1	≤0.5	≤0.25	≤1	>4/76	0.190657
A212	Shandong	M	69	Abdominal fluid	ST111	K63		4	≤8	16	≤4	2	>32	2	≤0.25	4	>4/76	0.412057
A141	Shandong	M	64	Sputum	ST15	K112		8	≤8	8	≤4	16	>32	>4	≤0.25	4	≤1/19	2.899057
A144	Hubei	F	1	Urine	ST2459	K15		16	≤8	≤1	≤4	≤1	≤1	1	≤0.25	2	>4/76	0.611657
A231	Shanghai	M	62	Pleural fluid	ST11	K64		16	>32	>16	>16	>32	>32	>4	>128	2	≤1/19	1.499457
A128	Jilin	F	55	Urine	ST15	K102		32	>32	>16	8	>32	>32	>4	≤0.25	2	>4/76	0.8732
A214	Hubei	M	58	bile	ST395	K48		32	>32	8	>16	>32	>32	>4	0.5	4	>4/76	1.2178
A218	Zhejiang	M	56	bile	ST3812	K107		32	≤8	>16	16	16	>32	2	4	≤1	≤1/19	0.493
A221	Zhejiang	F	66	Sputum	ST11	K64	iroB iucA rmpA rmpA2	64	≤8	>16	>16	32	>32	>4	>128	4	>4/76	0.7214
A224	Zhejiang	M	69	Blood	ST11	K64	iroB iucA	64	>32	>16	>16	>32	>32	>4	128	8	>4/76	0.3552
A225	Zhejiang	F	66	Sputum	ST11	K64	iroB iucA rmpA rmpA2	64	≤8	>16	>16	>32	>32	>4	>128	4	>4/76	0.7232
A140	Shandong	M	79	Sputum	ST86	K2	iroB iucA rmpA rmpA2 peg344	>64	≤8	≤1	≤4	≤1	≤1	≤0.5	≤0.25	2	≤1/19	0.0704
A30	Beijing	F	55	Urine	ST11	K47	iucA	>64	>32	>16	>16	>32	>32	>4	>128	2	>4/76	0.4552
A229	Zhejiang	F	47	Blood	ST11	K64		>64	≤8	>16	>16	>32	>32	>4	>128	2	>4/76	0.2916

Abbreviations: COL, colistin; AMK, Amikacin; FEP, Cefepime; FOX, Cefoxitin; CAZ, Ceftazidime; CRO, Ceftriaxone; CIP, Ciprofloxacin; ETP, Ertapenem; TGC, Tigecycline; SXT, Trimethoprim-Sulfamethoxazole

Figure 1. K. pneumoniae population structure and the prevalence of mcr gene and hvKp marker genes. The phylogeny in the left represents the population structure of 14 colistin-resistant isolated and 31 representative genomes of major global problem clones, with six hypervirulent clones (CG23, CG25, CG65, CG66, CG86, CG380) highlighted in blue branches, and 14 colistin-resistant isolated coloured in red text. Bootstrap values are also labelled on the nodes of the tree. The prevalence of mcr gene and hvKp marker genes are shown in the heatmap with whitespace separated. Different colors of the square in the heatmap represent the alignment identity for each gene (see the legend in the lower right section), with grey colour represents gene absence.

Figure 2. Relative expression of the pmrA, phoP, phoQ, mgrB, pmrC, and pmrK genes in colistin-resistant group and colistin-susceptible group. Values were represented by means of three independent replicates. Relative expression of pmrA, phoP, and phoQ in colistin-resistant group was significantly higher than colistin-susceptible group. There were 6 strains that less expressed mgrB gene, five strains harboured modified-mgrB gene and the rest strain were WT. However, because of early termination, overexpression of mgrB in one strain from ST11 could not form a protein with a negative regulatory function. *, P < 0.05; ns, not significantly different.

Table 2. Correlation coefficient between MIC and Chromosomal regulon gene expression.

			pmrA	phoQ	phoP	mgrB	pmrC	pmrK	MIC
Spearman's rho	pmrA	Correlation Coefficient	1	.759**	.571*	0.124	.405*	0.266	.471**
		Sig. (2-tailed)	.	0	0.021	0.484	0.017	0.128	0.005
	phoQ	Correlation Coefficient	.759**	1	.835**	-0.014	.435*	.510**	.766**
		Sig. (2-tailed)	0	.	0	0.937	0.01	0.002	0
	phoP	Correlation Coefficient	.571*	.835**	1	-0.269	.503*	0.487	.593*
		Sig. (2-tailed)	0.021	0	.	0.314	0.047	0.056	0.015
	mgrB	Correlation Coefficient	0.124	-0.014	-0.269	1	.382*	-0.121	-0.129
		Sig. (2-tailed)	0.484	0.937	0.314	.	0.026	0.495	0.468
	pmrC	Correlation Coefficient	.405*	.435*	.503*	.382*	1	.475**	0.224
		Sig. (2-tailed)	0.017	0.01	0.047	0.026	.	0.005	0.203
	pmrK	Correlation Coefficient	0.266	.510**	0.487	-0.121	.475**	1	.404*
		Sig. (2-tailed)	0.128	0.002	0.056	0.495	0.005	.	0.018
	MIC	Correlation Coefficient	.471**	.766**	.593*	-0.129	0.224	.404*	1
		Sig. (2-tailed)	0.005	0	0.015	0.468	0.203	0.018	.

** Correlation is significant at the 0.01 level (2-tailed). * Correlation is significant at the 0.05 level (2-tailed).

Figure 3. Transcriptional level and substitution mutations of chromosomal regulators in colistin-resistant isolates. The top six bar plots show the transcriptional level of six chromosomal regulators, with the x-axis represents 14 colistin-resistant isolates, ordered according to their colistin MICs, see the colour scale under the bar plot of pmrK. The prevalence of substitution mutations identified in nine chromosomal regulators are displayed in the heatmap following the colistin MICs. Each row shows a mutation site of corresponding gene (see the rightmost text), with mutated isolated colored in purple and those with no corresponding variation in grey. Genes absent in isolates are colored in white. Mutation reported in previous studies are marked with * in the rightmost. Three newly identified mutations which we speculated might have relation to colistin resistance are highlighted in red text.

Table 3. Colistin-resistant genes mediated by plasmid and mutations in the chromosomal genes in K. pneumoniae.

Name	COL mg/L	mcr	Amino acid alterations
			phoQ	phoP	pmrD	pmrK	pmrB	pmrA	pmrC	mgrB	CrrB	CrrA
A155	4	-	D150G	WT	D80G	D441E R372K R157S I117V	WT	WT	V39F C27F	WT	C68S	+
A212	4	mcr-1.1	D150G	WT	D80G	R157S Q156H	WT	WT	D477E A435T Q319R R152H V39F	WT	A43G I66V C68S Q239H T276A Q287K	+
A141	8	mcr-1.1	D150G	WT	D80G	R372K R157S Q156H	A246T	WT	S257L V39F	WT
A144	16	mcr-8.2	D150G	WT	D80G Q9K	R157S	A246T	S204L	V39F	WT	C68S	+
A231	16	-	D150G	WT	D80G	R372K R157S Q156H	R256G	WT	Q319R V39F C27F	WT
A128	32	-	D150G	WT	X82 D80G	R372K R157S	R256G	WT	Q319R V39F C27F	WT	C68S E189K	+
A214	32	-	D150G	WT	D80G	R372K R157S Q156H	A246T	WT	Q319R S257L V39F	M27K
A218	32	-	D150G	WT	D80G	R372K R157S Q156H	M285L	WT	Q319R R152H V39F	+
A221	64	-	D150G	WT	D80G	R372K R157S Q156H	R256G	WT	Q319R V39F C27F	ISKpn26
A224	64	-	D150G	WT	D80G	R372K R157S Q156H	R256G	WT	Q319R V39F C27F	ISKpn26
A225	64	-	D150G	WT	D80G	R372K R157S Q156H	R256G	WT	Q319R V39F C27F	ISKpn26
A140	>64	-	D150G	WT	D80G	R157S Q156H	WT	WT	V39F	WT	C68S	M1I S6T A94V
A30	>64	-	D150G	WT	D80G	R372K R157S Q156H	D313N R256G	WT	Q319R V39F C27F	Q30Stop
A229	>64	-	D150G	WT	D80G	R372K R157S Q156H	R256G	WT	Q319R V39F C27F	+

Abbreviations: COL: colistin; WT: wild-type.

Annotations of Amino acid alterations were based on the protein sequence of Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044 (Accession no. NC_012731.1), except that CrrA, CrrB were based on the protein sequence of Klebsiella pneumoniae subsp. pneumoniae MGH 78578 (Accession no. NC_009648.1).

The bold mutations were this article first found and reported, and the red mutation was that functional confirmation showed D313N could elevate the MIC of colistin.

Figure 4. Survival curve of six weeks CD1 mice after IP infection with colistin-resistance K. pneumoniae. Each group, with five mice included with one K. pneumoniae strain, either 5×10⁷ CFU of 14 CoRKp strains or NTUH-K2044, or 1×10⁸ CFU of low-virulent control QD110. Strain of ST86-K2 presented the highest level of virulence, more virulent than the reference strain NTUH-K2044, and six strains from ST11-K64/K47 showed intermediate virulence, except for ST111-K63 isolate, which showed weak virulenceto some extent. Isolate from ST5253-K28 was classic virulence. Data of classic-virulent strains from, with no death of mice occurring within 7 days, were not shown, as the survival consistent with QD110. P-values from the log-rank (Mantel-Cox) test were indicated as follows: *, P < 0.05; **, P <0.01; ns, not significantly different.

Figure 5. Differently expressed genes between colistin-resistant and colistin-sensitive isolates. (A) Volcano plots of the differently expressed genes. (B) Point plot of KEGG pathways for up-regulated genes. (C) Point plot of KEGG pathways for down-regulated genes.

Data availability

The raw data of both whole-genome DNA and RNA sequencing data have been deposited in the NCBI Sequence Read Archive under BioProject ID PRJNA762527.