High plasmid variability, and the presence of IncFIB, IncQ, IncA/C, IncHI1B, and IncL/M in clinical isolates of Klebsiella pneumoniae with bla KPC and bla NDM from patients at a public hospital in Brazil.

Oliveira, Érica Maria de; Beltrão, Elizabeth Maria Bispo; Scavuzzi, Alexsandra Maria Lima; Barros, Josineide Ferreira; Lopes, Ana Catarina Souza

doi:10.1590/0037-8682-0397-2020

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Revista da Sociedade Brasileira de Medicina Tropical

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Sumário

Major Article • Rev. Soc. Bras. Med. Trop. 53 • 2020 • https://doi.org/10.1590/0037-8682-0397-2020 copy

High plasmid variability, and the presence of IncFIB, IncQ, IncA/C, IncHI1B, and IncL/M in clinical isolates of Klebsiella pneumoniae with bla _KPC and bla _NDM from patients at a public hospital in Brazil.

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

INTRODUCTION

Antibiotic resistance in carbapenemase-producing Klebsiella pneumoniae is acquired and disseminated mainly by plasmids. Therefore, we aimed to investigate the occurrence of carbapenemase genes, analyze the genetic diversity by ERIC-PCR, and examine the most common plasmid incompatibility groups (Incs) in clinical isolates of K. pneumoniae from colonization and infection in patients from a hospital in Brazil.

METHODS

Twenty-seven isolates of carbapenem-resistant K. pneumoniae were selected and screened for the presence of carbapenemase genes and Incs by PCR, followed by amplicon sequencing.

RESULTS

The bla _KPC and bla _NDM genes were detected in 24 (88.8 %) and 16 (59.2 %) of the isolates, respectively. Thirteen isolates (48.1 %) were positive for both genes. The IncFIB (92.6 %) and IncQ (88.8 %) were the most frequent plasmids, followed by IncA/C, IncHI1B, and IncL/M, indicating that plasmid variability existed in these isolates. To our knowledge, this is the first report of IncHI1B in Brazil. We found eight isolates with clonal relationship distributed in different sectors of the hospital.

CONCLUSIONS

The accumulation of resistance determinants, the variability of plasmid Incs, and the clonal dissemination detected in K. pneumoniae isolates demonstrate their potential for infection, colonization, and the dissemination of different resistance genes and plasmids.

Keywords:
Klebsiella pneumoniae; Antimicrobial resistance; Plasmids; Incompatibility groups

Gene	Primer	Sequence (5`- 3`)	Temp.^a	Amplicon size (base pair)	Reference
bla _KPC	KPC-1a	TGTCACTGTATCGCCGTC	63°C	800	Yigit et al. (2001)⁴²42. Yigit H, Queenan AM, Anderson GJ, Sanchez AD, Biddle JW, Steward CD, et al. Novel carbapenem-hydrolyzing β-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob Agents Chemother . 2001;45(4):1151-61.
	KPC-1b	CTCAGTGCTCTACAGAAAACC
bla _GES	GES-F	GAAACCAAACGGGAGACGC	60ºC	207	Nordmann (2011)⁴³43. Nordmann P, Naas T, Poirel L. Global Spread of Carbapenemase producing Enterobacteriaceae. Emerg Infect Dis. 2011;17(10):1791-8.
	GES-R	CTTGACCGACAGAGGCAACT
bla _NDM	NDM-F	TAAAATACCTTGAGCGGGC	52ºC	439	Nordmann (2011)⁴³43. Nordmann P, Naas T, Poirel L. Global Spread of Carbapenemase producing Enterobacteriaceae. Emerg Infect Dis. 2011;17(10):1791-8.
	NDM-R	AAATGGAAACTGGCGACC
bla _VIM	VIM-F	CAGATTGCCGATGGTGTTTGG	62°C	600	Dong et al. (2008)⁴⁴44. Dong F, Xu XW, Song WQ, Lu P, Yu SJ, Yang YH, et al. Characterization of multidrug-resistant and metallo-betalactamase-producingPseudomonas aeruginosaisolates from a paediatric clinic in China. Chin Med J. 2008;121:1611-6.
	VIM-R	AGG TGGGCCATTCAGCCAGA
bla _IMP	IMP-F	GGAATAGAGTGGCTTAATTCTC	60°C	232	Dong et al. (2008)⁴⁴44. Dong F, Xu XW, Song WQ, Lu P, Yu SJ, Yang YH, et al. Characterization of multidrug-resistant and metallo-betalactamase-producingPseudomonas aeruginosaisolates from a paediatric clinic in China. Chin Med J. 2008;121:1611-6.
	IMP-R	GTGATGCGTCYCCAAYTTCACT
HI-2	IncHI-2-F	GGAGCGATGGATTACTTCAGTAC	64ºC	644	Caratolli et al. (2005)²⁸28. Caratolli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods. 2005;63:219-28.
	IncHI-2-R	GGCTCACTACCGTTGTCATCCT
L/M	IncL/M-F	GGATGAAAACTATCAGCATCTGAAG	62ºC	758	Caratolli et al. (2005)²⁸28. Caratolli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods. 2005;63:219-28.
	IncL/M-R	CTGCAGGGGCGATTCTTTAGG
A/C	IncA/C-F	GAGAACCAAAGACAAAGACCTGGA	62ºC	465	Caratolli et al. (2005)²⁸28. Caratolli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods. 2005;63:219-28.
	IncA/C-R	ACGACAAACCTGAATTGCCTCCTT
N	IncN-F	GTCTAACGAGCTTACCGAAG	62ºC	559	Caratolli et al. (2005)²⁸28. Caratolli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods. 2005;63:219-28.
	IncN-R	GTTTCAACTCTGCCAAGTTC
HI1B	HI1B-Fw	CAA AAC GAG AGA TAT TCAACCC CTG ATT	63ºC	900	Caratolli et al. (2005)²⁸28. Caratolli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods. 2005;63:219-28.
	HI1B-Rw	CTT GAT GAT ACA GGG
FIB	IncFIB-F	GGAGTTCTGACACACGATTTTCTG	62ºC	702	Caratolli et al. (2005)²⁸28. Caratolli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods. 2005;63:219-28.
	IncFIB-R	CTCCCGTCGCTTCAGGGCATT
Q	Ori-V-F	CTCCCGTACTAACTGTCACG	61ºC	436	Smalla et al. (2001)²⁷27. Smalla K, Heuer H, Gotz A, Niemeyer D, Krogerrecklenfort E, Tietze E. et al. Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids. Appl Environ Microbiol. 2000;66(11)4854-62.
	Ori-V-R	ATCGACCGAGACAGGCCCTGC
	Rep-B-F	TCGTGGTCGCGTTCAAGGTACG	64ºC	1.160
	Rep-B-R	CTGTAAGTCGATGATCTGGGCGTT
	Ori-T-F	TTCGCGCTCGTTGTTCTTCGAGC	63ºC	191
	Ori-T-R	GCCGTTAGGCCAGTTTCTCG
NA	ERIC-1	ATGTAAGCTCCTGGGGATTAAC	36°C	NA	Duan et al., (2009)²⁹29. Duan H, Chai T, Liu J, Zhang X, Qi C, Gao J, et al. Source identification of airborne Escherichia coli of swine house surroundings using ERIC-PCR and REP-PCR. Environ Res. 2009;109(5)511-7.
	ERIC-2	AAGTAAGTGACTGGGGTGAGCG

Isolates	Sector	Clinical sample	MIC(ERT)	MIC(IMI)	MIC(MER)	Resistance Genes	Incs	ERIC-PCR
K5-A3	ICU	Surgical drain	>4(R)	<=1(S)	2(I)	bla _KPC	Q, FIB	E7
K6-A3	CU	Blood	>4(R)	2(I)	4(R)	bla _KPC	Q, FIB	E2
K8-A3	ICU	Urine	>4(R)	>8(R)	>8(R)	bla _KPC, bla _NDM	Q, FIB	E2
K9-A3	GE	Blood	>4(R)	>8(R)	>8(R)	bla _KPC, bla _NDM	Q, FIB	E7
K10-A3	ICU	Blood	>4(R)	>8(R)	>8(R)	bla _KPC	Q, FIB, HI1B	E4
K11-A3	CU1	Blood	>4(R)	8(R)	4(R)	bla _KPC	Q, HI1B	E12
K12-A3	ICU	Blood	>4(R)	>8(R)	>8(R)	bla _KPC, bla _NDM	Q, FIB	E1
K16-A3	Cardiology	Urine	>4(R)	>8(R)	>8(R)	bla _NDM	FIB	E3
K24-A3	ICU	Urine	>4(R)	>8(R)	>8(R)	bla _KPC, bla _NDM	Q, FIB, A/C	E1
K26-A3	GE	Urine	>4(R)	8(R)	4(R)	bla _KPC, bla _NDM	Q, FIB	E11
K27-A3	MC	Urine	>4(R)	>8(R)	>8(R)	bla _KPC, bla _NDM	Q, FIB	E1
K30-A3	CU1	Blood	>4(R)	>8(R)	>8(R)	bla _KPC, bla _NDM	Q, FIB	E1
K31-A3	ICU	Urine	>4(R)	>8(R)	>8(R)	bla _KPC	FIB, A/C	E1a
K2-A3	Cardiology	Rectal Swab	>4(R)	(R)	(R)	bla _KPC	Q, FIB, L/M	E5
K3-A3	CU2	Rectal Swab	>4(R)	>8(R)	>8(R)	bla _KPC, bla _NDM	Q, FIB	E7
K13-A3	CU1	Rectal Swab	>4(R)	>8(R)	>8(R)	bla _KPC	Q, FIB, A/C	E10
K14-A3	CU2	Rectal Swab	>1(R)	>8(R)	>32(R)	bla _KPC	Q, FIB, HI1B	E4
K15-A3	Cardiology	Rectal Swab	>1(R)	>8(R)	>32(R)	bla _KPC	Q, FIB	E6
K17-A3	ICU	Rectal Swab	>4(R)	>8(R)	>8(R)	bla _KPC, bla _NDM	Q	E2
K18-A3	CU1	Rectal Swab	>4(R)	>8(R)	>8(R)	bla _KPC	Q, FIB	E1
K20-A3	CU1	Rectal Swab	>4(R)	>8(R)	>8(R)	bla _NDM	FIB	E1
K21-A3	Cardiology	Rectal Swab	>4(R)	>8(R)	>8(R)	bla _KPC	Q, FIB, A/C	E1a
K29-A3	CU	Rectal Swab	>4(R)	>8(R)	>8(R)	bla _KPC, bla _NDM	Q, FIB	E1
K32-A3	ICU	Rectal Swab	>4(R)	>8(R)	>8(R)	bla _KPC, bla _NDM	Q, FIB	E8
K34-A3	ICU	Rectal Swab	>4(R)	>8(R)	>8(R)	bla _NDM	Q, FIB	E1a
K36-A3	CU2	Rectal Swab	>4(R)	>8(R)	>8(R)	bla _KPC, bla _NDM	Q, FIB, L/M	E9
K37-A3	ICU	Rectal Swab	>4(R)	>8(R)	>8(R)	bla _KPC, bla _NDM	Q, FIB	E1

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[1] Corresponding author: Dra. Ana Catarina de Souza Lopes. e-mail:ana.lopes.ufpe@gmail.com