The hypermucoviscosity of hypervirulent K. pneumoniae confers the ability to evade neutrophil-mediated phagocytosis

Figures & data

Table 1. Phenotypic and genotypic characteristics of K. pneumoniae strains used in this study

Strain Name	Strain ID used in this paper		STs	MIC [µg mL^−1]												rmpA	rmpA2	String Test
				MEM		CTX		CAZ		AMK		CIP	TIG	PB	TE
17ZR-101		HvKP1	ST86		32		> 128		> 128		4	4	0.25	4	> 128	+	truncated	+
HvKP1088[38]		HvKP2	ST23		64		< 0.06		128		2	64	0.5	2	> 128	+	truncated	+
GH22		HvKP3	ST23		< 0.06		< 0.06		2		2	< 0.06	0.25	4	> 128	+	truncated	+
EH78		HvKP4	ST23		< 0.06		< 0.06		0.25		1	< 0.06	0.25	4	> 128	+	truncated	+
17ZR-66		HvKP5	ST11		64		> 128		> 128		> 128	> 32	0.5	1	128	truncated	+	-
FJ8[39]		cKP1	ST11		32		64		> 128		128	64	1	4	2	-	-	-
HKU1		cKP2	ST716		0.06		> 128		8		128	8	4	2	4	-	-	-
KP04-1		cKP3	ST11		0.06		128		128		64	32	4	4	16	-	-	-
PM-28		cKP4	ST14		2		4		16		2	1	0.5	2	32	-	-	-
16HN-35		NP-HvKP	ST11		> 128		> 128		> 128		2	64	0.5	2	> 128	truncated	truncated	-
16HN-35/A		NP-HvKP /A	ST11		> 128		> 128		> 128		2	64	0.5	2	> 128	+	-	+
16HN-35/A2		NP-HvKP /A2	ST11		> 128		> 128		> 128		4	> 64	0.5	2	> 128	-	+	+
WZ1-2[9,10]		NP-HvKP-PC	ST11		64		> 128		32		2	64	0.5	2	0.5	-	-	-
WZ1-2-TC[9,10]		NP-HvKP-TC	ST11		>128		> 128		> 128		1	> 128	0.5	2	> 128	+	-	+

MEM, meropenem; CTX, cefotaxime; CAZ, ceftazidime; AMK, amikacin; CIP, ciprofloxacin; TIG, tigecycline; PB, polymyxinB(E); TE, tellurite.

“ – ” indicates that the experiment or analysis was not applicable to this strain.

Figure 1. The hypermucoid phenotype of K. pneumoniae is associated with inhibition of cell adherence and internalization. Mucoviscosity (a) and uronic acid production (b) in hypervirulent Klebsiella strains HvKP1, HvKP2, HvKP3, HvKP4, and classical Klebsiella strains cKP1, cKP2, cKP3, cKP4. Invasion assay of selected clinical K. pneumoniae strains using Macrophage RAW 264.7 cells (c) and intestinal epithelial Caco-2 cells (d). Adhesion assay of selected strains using lung epithelial A549 cells (e) and Caco-2 cells (f). Data were analyzed by one-way ANOVA test. Each data point was repeated three times (n = 3). Data are presented as the mean ± s.e.m. * P < 0.05; ** P < 0.01; ***P < 0.001; **** P < 0.0001. Abbreviation: NS, not significant

Figure 2. Cell adherence and internalization assay of K. pneumoniae strain NP-HvKP variants and HvKP5. Mucoviscosity (a), and uronic acid production (b) of Klebsiella strain NP-HvKP, NP-HvKP/A, NP-HvKP/A2, NP-HvKP-PC, NP-HvKP-TC and HvKP5. Assay of the ability of selected strains to adhere to lung epithelial A549 cells (c) and Caco-2 cells (d). Invasion assay of selected clinical K. pneumoniae strains using Macrophage RAW 264.7 cells (e) and intestinal epithelial Caco-2 cells (f). Data were analyzed by one-way ANOVA test. Each data point was repeated three times (n = 3). Data are presented as the mean ± s.e.m. * P < 0.05; ** P < 0.01; **** P < 0.0001. Abbreviation: NS, not significant

Figure 3. HvKP strains escape neutrophil-mediated phagocytosis and killing. (a) Neutrophil-like cell dHL-60 phagocytosis assay of HvKP1, HvKP2, cKP2, NP-HvKP, NP-HvKP/A, and NP-HvKP/A2. (b) Neutrophil killing assays of Klebsiella pneumoniae strains. (c) The resistance of K. pneumoniae strains to killing by pooled human serum. (d) Bacteria loads in blood, liver, lungs, kidney, and spleen were measured by plating at 24 hr post-infection. Bacterial load detectable in blood and various organs: HvKP1, cKP2, NP-HvKP/A and NP-HvKP. Data were analyzed by one-way ANOVA test. Each data point was repeated three times (n = 3). Data are presented as the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001

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