Prevalence of hypervirulent and carbapenem-resistant Klebsiella pneumoniae under divergent evolutionary patterns

Figures & data

Figure 1. The prevalence of hv-CRKP and CR-hvKp strains. Distribution of carbapenemases is depicted as below. Intensity of box shading indicates the proportion of genomes harbouring different carbapenemases (orange), as per inset legend.

Figure 2. The mobilization of non-conjugative virulence plasmids by conjugative KPC plasmid. (A) Two rounds plasmid conjugation experiments simulating the transfer path of non-conjugative plasmid. (B) The confirmation of transconjugants by PFGE and S1-PFGE. *Virulence plasmid pK2044. ^#KPC plasmid pKPHS2. The same symbol is used to represent the parental strain and its transconjugant.

Figure 3. The transfer potential of mobilizable virulence plasmids. (A) The mobilization of mimic virulence plasmid by conjugative KPC plasmid pKPHS2. (B) The distribution of TraM_pKPHS2-carrying plasmids in Enterobacteriaceae. (C) nic sites of pKPHS2 and pK2044. Alignment was generated with the MUSCLE sequence alignment tool.

Table 1. Antimicrobial susceptibilities of strains and their transconjugants.

Strains	STs	Bacterial species	MIC (µg/mL)^a
			AMK	SAM	TZP	CZO	CXM	CRO	FEP	CSL	CHL	GEN	LVX	SXT	MEM	IPM	TGC	NIT
NTUH-K2044	ST23	K. pneumoniae	<8	8/4	<8/4	1	16	<0.25	<2	<4/2	32	<1	<0.5	<0.25/4.75	<0.25	<0.25	1	64
HS11286	ST11	K. pneumoniae	>128	>64/32	>256/4	>16	>64	>8	16	128/64	>64	>32	2	>8/152	32	64	<0.5	256
JS187	ST11	K. pneumoniae	>128	>64/32	>256/4	>16	>64	>8	>64	128/64	>64	>32	8	>8/152	64	64	<0.5	256
J53	-	E. coli	<8	4/2	<8/4	1	8	<0.25	<2	<4/2	8	<1	<0.5	<0.25/4.75	<0.25	<0.25	<0.5	<8
M-NTUH-K2044-pKPSH2^b	ST23	K. pneumoniae	<8	>64/32	32/4	>16	>64	>8	<2	8/16	16	<1	<0.5	<0.25/4.75	2	2	<0.5	64
NM-NTUH-K2044-pKPSH2^c	ST23	K. pneumoniae	<8	>64/32	>256/4	>16	>64	>8	<2	32/16	8	<1	<0.5	<0.25/4.75	16	32	<0.5	32
JS187-pK2044	ST11	K. pneumoniae	>128	>64/32	>256/4	>16	>64	>8	>64	>128/64	>64	>32	8	>8/152	64	64	<0.5	>256
J53-pK2044	-	K. pneumoniae	8	>64/32	32/4	>16	>16	>64	<2	16/8	8	<1	<0.5	<0.25/4.75	1	1	<0.5	<8

^a AMK, amikacin; SAM, ampicillin/sulbactam; TZP, piperacillin/tazobactam; CZO, cefazolin、CXM, cefuroxime; CRO, ceftriaxone; FEP, cefepime; CSL, cefoperazone/sulbactam; CHL, chloramphenicol; GEN, gentamicin; LVX, levofloxacin; SXT, trimethoprim/sulphamethoxazole; MEM, meropenem; TGC, tigecycline; NIT, nitrofurantion.

^b Three M transconjugants were respectively selected in 1st, 12th, and 24th passage for antimicrobial susceptibility tests. The results were combined together due to the same resistance phenotype.

^c Three NM transconjugants were respectively selected in 1st, 12th, and 24th passage for antimicrobial susceptibility tests. The results were combined together due to the same resistance phenotype.

Figure 4. The virulence phenotypes and levels of CR-hvKp strain. (A) Mucoviscosity. (B) The relative expression of capsular synthesis genes. (C) Uronic acid. (D) Transmission electron microscopy of the capsule. (E) Biofilm formation. (F) Siderophores production. (G) Serum resistance. (H) The survival curves of infected Larvae wax. (I) The survival curves of infected mice. An unpaired two-sided Student’s t-test was performed for mucoviscosity, uronic acid, biofilm formation, siderophores production, and serum resistance. A log-rank (Mantel–Cox) test was performed for the survival curves. ***P< 0.001, **P< 0.01, *P< 0.05, ns: not significant.

Figure 5. The influence of rfaH to carbapenem resistance and CPS production.

Figure 6. The virulence phenotypes and levels of hv-CRKP. (A) Growth curve. (B) Siderophores production quantification. (C) Siderophores production determined by CAS agar plate. (D) Mucoviscosity. (E) Transmission electron microscopy of the capsule. (F) Uronic acid. (G) Biofilm formation. (H) Serum resistance. (I) The survival curves of infected mice. (J) The survival curves of infected larvae wax. An unpaired two-sided Student’s t-test was performed for mucoviscosity, uronic acid, biofilm formation, siderophores production, and serum resistance. A log-rank (Mantel–Cox) test was performed for the survival curves. ****P < 0.0001, ***P< 0.001, **P< 0.01, *P< 0.05, ns: not significant.