Emergence of NDM-5-Producing Carbapenem-Resistant Klebsiella pneumoniae and SIM-Producing Hypervirulent Klebsiella pneumoniae Isolated from Aseptic Body Fluid in a Large Tertiary Hospital, 2017–2018: Genetic Traits of blaNDM-Like and blaSIM-Like Genes as Determined by NGS

Figures & data

Table 1 Clinical Characteristics of Patients Infected with CRKP and hvKP

Isolates	Sex	Age	Unit	Date of Hospitalization	LOS	Date of Specimen	Specimen	Outcome
CRKP58	F	56	Hematopathology	2017.9.28–2018.1.7	101	1.5	bl	Dead
hvKP75	M	48	Emergency	2018.2.14	1	2.14	hydropericardium	Give up treatment
CRKP87	M	76	Neurosurgery	2018.3.29–2018.4.28	30	4.25	scf	Dead
CRKP90	M	62	ICU	2018.4.16–2018.6.12	57	5.8	bl	Discharge
CRKP93	M	62	Rheumatology	2018.5.9–2018.5.30	21	5.21	bl	Discharge
CRKP95	M	44	General surgery	2018.4.1–2018.6.1	61	5.16	puncture fluid	Discharge
CRKP96	M	42	ICU	2018.6.7–2018.6.29	22	6.27	bl	Discharge
CRKP106	F	44	Hematopathology	2018.6.28–2018.8.1	34	7.31	bl	Dead
CRKP110	F	76	ICU	2018.7.17–2018.9.1	46	8.8	scf	Give up treatment
CRKP112	M	45	ICU	2018.7.28–2018.9.25	59	8.17	bl	Discharge
CRKP114	F	68	Hematopathology	2018.8.22–2018.9.29	38	9.26	bl	Dead
CRKP121	F	85	ICU	2018.11.5–2018.12.14	39	11.20	pus	Dead

Abbreviations: M, male; F, female; ICU, intensive care unit; LOS, length of hospital stay; bl, blood; scf, cerebrospinal fluid.

Table 2 Demographics of Patients Infected with CRKP and hvKP

Characteristics	No. (%) of Patients
	CRKP (N = 11)	hvKP (N = 1)
Demographics
Infection sites, no. (%)
Pneumonia infection	6 (54.5%)
Bloodstream infection	4 (36.4%)
Catheter related bloodstream infection	2 (18.2%)
Intracranial infection	2 (18.2%)
Intra-abdominal infection	1 (9.1%)
Pus infection	1 (9.1%)
Hydropericardium infection		1 (100%)
Underlying diseases, no. (%)
Diabetes	2 (18.2%)
Hypertension	2 (18.2%)
Alcohol history	2 (18.2%)
Smoking history	5 (45.5%)
Cardiovascular disease	2 (18.2%)
Leukemia	3 (27.3%)
Liver disease		1 (100%)
Immune deficiency	9 (81.8%)
Invasive procedures during hospitalization, no. (%)
Thoracentesis	3 (27.3%)
Mechanical ventilation	6 (54.5%)	1 (100%)
Central intravenous catheter	8 (72.7%)
Urinary catheter	9 (81.8%)
Tracheal cannula	6 (54.5%)
Tracheotomy	3 (27.3%)
Nasal catheter	1 (9.1%)
Stomach tube	5 (45.5%)
ICU stay no. (%)	7 (63.6%)
Outcomes no. (%)
Death^a	7 (58.3%)

Note: ^aThe mortality of this study including the patients who abandoned treatment but had specific indicators of death, such as deep shock, dilation of the pupils, and cervical pulse disappeared.

Figure 1 Distribution of therapeutic regimens of patients with CRKP infection.

Table 3 Antimicrobial Susceptibility Testing Results of 12 K. pneumoniae Isolates (Mg/L)

Isolates	Carbapenem			Cephalosporins				Monobactams	β-lactam		Quinolones		Tetracycline	Fosfomycin	Chloramphenicol	Aminoglycoside
	MEM	IPM	ETP	CRO	CAZ	CTX	FEP	ATM	CSL	TZP	CIP	LEV	TGC	FOS	CHL	AMK
CRKP58	1	4	0.5	256	32	>256	>256	128	128	32	>64	32	16	128	128	4
hvKP75	0.032	0.25	≤0.016	≤0.5	≤0.5	<0.5	≤0.5	≤0.5	<0.5	2	<0.016	0.064	8	16	16	1
CRKP87	32	8	32	>256	>256	>256	128	256	>256	>256	64	64	2	32	32	2
CRKP90	8	16	128	>256	128	>256	256	256	>256	>256	>64	64	2	128	64	4
CRKP93	32	8	16	>256	>256	>256	128	512	>256	>256	64	64	2	32	64	2
CRKP95	16	8	32	>256	>256	>256	64	256	>256	>256	64	64	2	16	64	2
CRKP96	64	8	64	≥256	>256	>256	128	512	>256	>256	>64	>64	16	16	128	1
CRKP106	16	32	16	>256	>256	>256	32	<0.25	>256	128	0.064	0.064	16	32	>256	2
CRKP110	64	32	128	≥256	64	256	64	512	>256	>256	64	32	2	256	32	1
CRKP112	16	16	128	>256	>256	>256	64	256	>256	>256	>64	64	2	128	64	4
CRKP114	4	1	2	>256	>256	>256	>256	>512	128	>256	1	2	32	>256	32	2
CRKP121	32	32	64	>256	>256	>256	128	512	>256	<0.5	>64	>64	32	32	128	1

Abbreviations: MEM, meropenem; IPM, imipenem; ETP, ertapenem; CRO ceftriaxone; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; ATM, aztreonam; CSL, cefoperazone/sulbactam; TZP, piperacillin/tazobactam; CIP, ciprofloxacin; LEV, levofloxacin; TGC, tigecycline; FOS, fosfomycin; CHL, chloramphenicol; AMK, amikacin.

Table 4 Distribution of Resistance Mechanism and Virulence Genes Harbored by CRKP and hvKP Strains

Isolates	MHT	mCIM/eCIM	Carbapenemase					Ampc			ESBLs			MCR	ST	String Test	Virulence-Associated Genes				Serotypes
			KPC	NDM	IMP	SIM	others	DHA	ACT	CMY	CTX-M-9,13-14,16-19,Toho-1	CTX-M-1,3,10-12,15	other				iucA	iutA	rmpA	rmpA2	iroN	wzi	KL-type
CRKP58	−	I/－	－	－	－	－	－	－	－	－	－	CTX-M-3	－	－	ST147	－	－	－	－	－	－	K41	KL106
hvKP75	−	－/－	－	－	－	SIM	－	－	－	－	－	－	－	－	ST1764	＋	－	－	＋	＋	＋	K64	KL64
CRKP87	−	+/+	－	NDM-1	－	－	－	－	－	－	－	－	－	－	ST11	－	－	－	－	－	－	─	KL22KL37
CRKP90	−	+/+	－	－	－	－	－	－	－	－	－	－	－	－	ST11	－	－	－	－	－	－	─	KL22KL37
CRKP93	−	+/+	－	－	－	－	－	－	－	－	－	－	－	－	ST11	－	－	－	－	－	－	─	KL22KL37
CRKP95	−	+/+	－	－	－	－	－	－	－	－	－	－	－	－	ST11	－	－	－	＋	＋	＋	─	KL22KL37
CRKP96	−	+/+	－	NDM-1	－	－	－	－	－	－	－	－	－	－	ST11	－	－	－	－	－	－	─	KL22KL37
CRKP106	−	+/+	－	NDM-5	－	SIM	－	－	－	－	－	－	－	－	ST690	－	－	－	－	－	－	─	─
CRKP110	−	+/−	KPC-2	－	－	－	－	－	－	－	CTX-M-14	－	－	－	ST11	－	－	－	－	－	－	K47	KL47
CRKP112	−	+/+	－	NDM-1	－	－	－	－	－	－	－	－	－	－	ST11	－	－	－	－	－	－	─	KL22KL37
CRKP114	−	－/－	－	－	－	－	－	－	－	－	－	－	－	－	ST7	－	－	－	－	－	－	─	KL54
CRKP121	−	+/+	－	－	－	－	－	－	－	－	－	－	－	－	ST524	－	－	－	－	－	－	─	KL22KL37

Notes: ⁺Indicates the presence of the corresponding gene or positive for this test; ^–Indicates the absence of the corresponding gene or negative for this test.

Figure 2 Distribution of MICs with NDM-1, NDM-5 and KPC-2/CTX-M-14.

Notes: MIC distribution of ATM, quinolones, and TGC in bla_NDM-1 (n = 3), bla_NDM-5 (n =1), and bla_{KPC-2/CTX-M-14} (n = 1) CRKP strains.

Figure 3 Molecular phylogenetic analysis of 12 isolates.

Notes: The evolutionary history by using maximum likelihood method based on the Kimura-2-parameter model; evolutionary analyses were conducted in MEGA-X.

Table 5 The Distribution of Resistance Genes

Drug Class	Target	CTX-M-3 ST147 CRKP58	NDM-1 ST11 CRKP112	SIM ST1764 hvKP75
Cephalosporin	CTX-M-3	＋	＋
	CTX-M-14
	CTX-M-17
Fluoroquinolones	oqxA	＋	＋	＋
	oqxB	＋	＋
	QnrB1	＋
	QnrB13	＋
	QnrB15	＋
	QnrB2	＋
	QnrB23	＋
	QnrB26	＋
	QnrB3	＋
	QnrB30	＋
	QnrB5	＋
	QnrB6	＋
	QnrB7	＋
	QnrS1	＋	＋
	QnrS4	＋	＋
	QnrS6	＋	＋
	emrB			＋
Carbapenems	NDM-1		＋
	NDM-10		＋
	NDM-4		＋
	NDM-5		＋
	NDM-6		＋
	NDM-7		＋
	NDM-9		＋
ESBLs	SHV-187		＋
	SHV-71			＋
Aminoglycosides	APH(3ʹ)-Ia
	APH(3ʹ’)-Ib
	APH(6)-Id
	AAC(3)-IIb	＋	＋
	aadA6/aadA10	＋
	abeM		＋
	adeB		＋
	adeF			＋
	adeG-L		＋
	adeR		＋
	bacA			＋
Antibiotic efflux	baeR			＋
Chloramphenicol	floR	＋
Fosfomycin	FosA2			＋
	H-NS			＋
	Klebsiella			＋
	marA			＋
	mdtC			＋
Macrolides	mphA	＋	＋
	Mrx	＋	＋
	vgaC			＋
	rmtB
	ramA			＋
	PC1		＋
Plasmid partition protein A	patA			＋
Tetracycline	tet(C)	＋

Table 6 The Distribution of Virulence Genes

Virulence Class	Target	CTX-M-3 ST147 CRKP58	NDM-1 ST11 CRKP112	SIM ST1764 hvKP75
Lipopolysaccharide	O-antigen export system ATP-binding protein	＋
	O-antigen export system permease protein RfbD	＋
	Kvar			＋
	cpsB			＋
	lpxA		＋
	lpxC		＋
	htrB		＋
Capsule	RcsAB	＋	＋	＋
	phosphomannomutase		＋
	cpsB mannose-1-phosphate guanylyltransferase		＋	＋
	uge	＋	＋	＋
	ugd			＋
	GalF	＋	＋	＋
Type-I fimbriae cluster	fimH	＋	＋
	fimA			＋
	fimB			＋
	fimC	＋
	fimE			＋
	fimF			＋
Type-III fimbriae cluster	mrkA	＋	＋	＋
	mrkB	＋	＋
	mrkC	＋	＋	＋
	mrkD	＋	＋
	mrkF	＋	＋	＋
	mrkI			＋
	magA
Aerobicactin	iutA	＋	＋
Iro	fepD	＋	＋	＋
	fepC			＋
Iuc	iucA			＋
	iucB			＋
	iucC			＋
	iucD			＋
Sal	IroE	＋	＋
	IroN	＋	＋
	fepA			＋
	rpoS			＋
Ent	entB			＋
	entC			＋
	entE			＋
	entF			＋
	entS	＋	＋
	fepA	＋	＋	＋
	entD	＋		＋
Pumps, Transports	RND efflux system	＋	＋
	fimD	＋	＋
	ribose ABC transport system		＋	＋
	ABC transporter		＋
	AdeFGH efflux pump/transport autoinducer		＋
Regulator	rmpA
	rmpA2			＋
	BfmRS		＋
	transcriptional regulator		＋
alls	all			＋
T6SS	clpB	＋	＋	＋
	kvar			＋
	TssF			＋
	TssM	＋
Other	acrA	＋	＋	＋
	acrB			＋
	pu1G			＋
	pu1I			＋
	pu1J			＋
	pu10	＋	＋	＋
	pu1S			＋
	pu1D	＋	＋
	pu1E	＋	＋

Table 7 The FICs of CRKP Isolates

Isolates	FIC
	TGC+IPM	TGC+MEM	TGC+ATM
CRKP58	0.0938	0.5313	0.0625
hvKP75	─	─	─
CRKP87	0.0938	0.0625	0.0625
CRKP90	0.0625	0.125	0.09375
CRKP93	0.0938	0.3475	0.0625
CRKP95	0.0938	0.0625	0.0625
CRKP96	0.0938	0.0938	0.125
CRKP106	0.0625	0.0625	─
CRKP110	0.0625	0.0625	0.1563
CRKP112	0.0625	0.0938	0.0625
CRKP114	0.0938	0.0938	0.0625
CRKP121	0.0625	0.0625	0.5313

Note: The FIC index was interpreted as ≤0.5, synergy, >0.5 to≤4, additive, and >4, antagonism.