F33:A−:B− and F2:A−:B− Plasmids Mediate Dissemination of rmtB-bla_CTX-M-9 Group Genes and rmtB-qepA in Enterobacteriaceae Isolates from Pets in China

Abstract

This study investigated the prevalence of 16S rRNA methylase genes in 267 Enterobacteriaceae isolates collected from pets. The rmtB gene was detected in 69 isolates, most of which were clonally unrelated. The coexistence of the rmtB gene with the bla_CTX-M-9 group genes and/or qepA within the same IncFII replicons was commonly detected. The two dominant types of IncF plasmids, F2:A−:B−, carrying rmtB-qepA, and F33:A−:B−, carrying the rmtB-bla_CTX-M-9 group genes (and especially bla_CTX-M-65), shared restriction patterns within each incompatibility group.

TEXT

Recently, the production of 16S rRNA methylases by Gram-negative bacilli has emerged as a novel mechanism for their high-level resistance to aminoglycosides (9, 31). Seven plasmid-mediated 16S rRNA methylase genes, rmtA, rmtB, rmtC, rmtD, rmtE, armA, and npmA, have been identified so far in multiple species of Enterobacteriaceae (3, 6, 9, 13, 31). These resistance determinants are globally disseminated, with rmtB and armA being the most frequently reported types worldwide (3, 5, 9, 10, 11, 13, 19, 27, 29, 32). However, even though aminoglycosides are widely used in pets to treat Gram-negative bacterial infections, the prevalence of 16S rRNA methylases in bacteria from pets is not known. In addition, the 16S rRNA methylase genes, especially rmtB, are commonly associated with bla_CTX-M genes (1–3, 8, 13, 19, 27, 29, 32). In our recent study on the distribution of extended-spectrum β-lactamase (ESBL) genes in Escherichia coli isolates obtained from pets, some CTX-M-producing isolates showed significantly reduced susceptibilities to amikacin, and this resistance to amikacin was cotransferred with CTX-M-9 subgroup genes to recipients (25). The current study investigated the prevalence of 16S rRNA methylase genes among CTX-M-producing isolates and characterized the plasmids carrying rmtB.

The present study included 135 CTX-M-producing Enterobacteriaceae isolates (119 E. coli, 11 Klebsiella pneumoniae, 3 Enterobacter cloacae, and 2 Citrobacter freundii isolates) recovered from healthy or diseased pets (dogs and cats) in Guangdong, China, during 2006 and 2008. The ESBL genes in most of these strains have been characterized previously (18, 25). The gene type of bla_CTX-M was confirmed by PCR and DNA sequencing (25). In addition, this study also included 132 Enterobacteriaceae isolates previously confirmed to be CTX-M negative that were collected from pets in Guangdong province of China during 2006 and 2008 (18, 25). The presence of 16S rRNA methylase genes was identified by PCR using previously designed primers (5, 6, 9). Of the 135 CTX-M producers, 60 (∼44%) were positive for rmtB and 5 (∼4%) were positive for armA (Table 1). No isolate was positive for the rmtA, rmtC, rmtD, rmtE, or npmA genes. The rmtB gene was also detected in 9 (∼7%) of the 132 CTX-M-negative isolates. Therefore, of the 69 rmtB-positive isolates, 60 were CTX-M producers, and most of the enzymes produced belonged to the CTX-M-9 group. Since the plasmid-mediated fluoroquinolone efflux pump gene qepA is frequently associated with rmtB (1, 2, 17, 22, 23, 28, 32), RmtB-producing isolates were screened for the qepA gene (17). Our results showed that 31 (44.9%) RmtB-producing isolates were positive for qepA. The pulsed-field gel electrophoresis analysis revealed that most of the rmtB-positive isolates were clonally unrelated (Table 1).

Table 1.

Distribution of 16S rRNA methylase genes among Enterobacteriaceae isolates and diversity of these isolates

CTX-M type(s) of β-lactamase (no. of isolates)	No. (%) of isolates positive for:			No. of PFGE subtypesb
	rmtB	armA	qepAa
CTX-M-9 type (88)	46 (52.3)	4	17 (37.0)
CTX-M-14 (57)	22	2	10	45 (4)
CTX-M-24 (13)	9		6	8 (1)
CTX-M-65 (9)	9		1	6 (1)
CTX-M-27 (8)	5	2		8
CTX-M-9 (1)	1			1
CTX-M-9 type and CTX-M-1 type (17)	10 (58.8)		7
CTX-M-14, CTX-M-82 (1)				1
CTX-M-24, CTX-M-82 (1)	1			1
CTX-M-14, CTX-M-55 (9)	5		5	7 (1)
CTX-M-65, CTX-M-55 (1)	1			1
CTX-M-14, CTX-M-64, CTX-M-55 (1)	1		1	1
CTX-M-14, CTX-M-64 (1)				1
CTX-M-27, CTX-M-64 (1)				1
CTX-M-14, CTX-M-3 (1)	1		1	1
CTX-M-9, CTX-M-55 (1)	1			1
Any CTX-M-1 type (30)	4 (13.3)	1	3
CTX-M-55 (17)	2		1	14 (2)
CTX-M-64 (1)				1
CTX-M-3 (6)	2	1	2	4 (2)
CTX-M-15 (6)				5
CTX-M-positive isolates (135)	60 (44.4)	5	27 (45.0)
CTX-M-negative isolates (132)	9 (6.8)		4
All isolates (267)	69 (25.8)	5	31 (44.9)

^aOnly rmtB-positive isolates were screened for the qepA gene.

^bThe number of nontypeable isolates is indicated in parentheses. PFGE, pulsed-field gel electrophoresis.

The transferability of the rmtB genes was studied by conjugation experiments as previously described (7). When plasmid cotransfer occurred, the transformation experiment was carried out. The presence of rmtB, qepA, and bla_CTX-M in the transconjugants and transformants was confirmed by PCR as previously described (5, 17). Of the 69 rmtB-positive isolates, 73 transconjugants/transformants containing rmtB were obtained from 66 isolates, with 6 donors generating two or three transconjugants carrying different plasmids and resistance genes. rmtB was cotransferred with bla_CTX-M or qepA genes in 33 and 25 transconjugants/transformants, respectively (Table 2). In addition, 8 transconjugants carried rmtB, qepA, and bla_CTX-M-9G simultaneously.

Table 2.

Replicon sequence typing of plasmids in transconjugants carrying rmtB

Resistance gene(s) in transconjugant (n)a	No. of plasmids with indicated replicon type(s)b
	N	N, F39	N, F2	F1	F2	F18	F33	F33, B1	F34	F35	F35, B20	F42	F22, B6	F36, B6	Unknown
rmtB (15)	1	1			3	1	2								7
rmtB, blaCTX-M-9G (22)					4		12	2					1		3
rmtB, blaCTX-M-1G (2)															2
rmtB, qepA (25)	1			1	22							1
rmtB, qepA, blaCTX-M-9G (8)			1		2				1	1	1			2
rmtB, qepA, blaCTX-M-1G (1)															1
Total (73)	2	1	1	1	31	1	14	2	1	1	1	1	1	2	13

^an, number of transconjugants carrying the indicated resistance gene(s).

^bN, IncN; F, IncFII allele; B, IncFIB allele.

PCR-based plasmid replicon typing was performed to characterize the conjugative plasmids carrying rmtB (4). The IncFII, IncFIB, and IncN replicon types were detected in 58 (79.5%), 5 (6.8%), and 4 (5.5%) of the plasmids from the 73 transconjugants, respectively, with 7 other transconjugants carrying two replicons (FII in combination with FIB or N) (Table 2). To better clarify the IncF plasmids, a replicon sequence typing scheme discriminating IncF plasmid variants, described by Villa et al. (26), was used to characterize the IncFII and IncFIB replicons. Among these transconjugants, 10 and 3 different alleles were identified for the FII and FIB replicons, respectively (Table 2). The F2 allele was detected in 24 out of the 34 transconjugants that carried both rmtB and qepA, and the F33 allele, a new F allele identified in this study, was detected in 12 out of the 22 transconjugants that carried both rmtB and bla_CTX-M-9G. Three transconjugants obtained from one E. coli donor (0113DDF) carried different IncF plasmids (p0113J, p0113-1, and p0113-2T) encoded by different resistance genes (Table 3), indicating the coexistence of three unrelated plasmids in one bacterium. It has been demonstrated that the IncF plasmids possess great versatility in intracellular adaptation due to the rapid evolution of the regulatory sequences of the replicons (21, 26). The coexistence and maintenance of three IncF plasmids that belong to different subgroups, F2:A−:B−, F35:A−:B−, and F33:A−:B−, in the same bacterial strain indicated preliminarily that these differences in the F alleles could result in the emergence of compatible plasmids.

Table 3.

Characterization of some IncF plasmids carrying rmtB^a

Plasmid(s)	Resistance gene(s)	Additional resistance(s)d	MIC of ciprofloxacin (μg/ml)	FAB formula or replicon typee	EcoRI plasmid RFLP	BamHI plasmid RFLP	Plasmid size (kb)
p4A2J, p2A11J, p4C7J, p3C6J, pLC57J, p1F12J, pLC71J, p3B6J, p1B4J, p1D1J, p1C1J, p3A11J, pLC73J,b p3C8J, p0113J, p2A8J, pLC55J	rmtB, qepA	SXT (4 strains)	0.06 to 0.25	F2:A−:B−	A1	1a	∼75
p3D10J	rmtB, qepA	SXT, TET	0.06	F2:A−:B−	B	1b	∼80
p4D8J	rmtB, qepA, blaCTX-M-14	CHL	0.125	F2:A−:B−	A1	1a	∼75
p1D5J	rmtB, blaCTX-M-65		0.008	F2:A−:B−	A3	1c	∼63
p4C12T	rmtB, qepA, blaCTX-M-14		0.125	F2:A−:B−	C	2	∼105
p2D11J	rmtB, qepA	SXT, TET, STR	0.06	F2:A−:B−	D	3	∼80
p7A1J	rmtB, blaCTX-M-27		0.008	F2:A−:B−	E	4	∼85
p0113-2T	rmtB, qepA, blaCTX-M-14	NAL, TET	0.06	F35:A−:B−	F	5	∼95
pLC23Tb	rmtB, qepA, blaCTX-M-24a	NAL, TET	0.06	F35:A−:B−	F	5	∼95
p2D2T	rmtB, qepA	NAL	0.5	F34:A−:B−	G	6	∼70
p3A9J	rmtB, qepA	SXT	0.06	F42:A−:B−	H	7	∼90
pLC40J	rmtB, qepA		0.25	N	I	8	∼45
p4B5J, p2B1J, p2F2J, p4A1J, p3D12T,c p7A8T	rmtB, blaCTX-M-65	SXT (4 strains)	0.008	F33:A−:B−	J1	9	∼65
p2C5J, p0113-1, pZ426-1	rmtB, blaCTX-M-14		0.015	F33:A−:B−	J1	9	∼65
p80111-3	rmtB, blaCTX-M-9	TET	0.015	F33:A−:B−	J2	9	∼65
pLC47J	rmtB		0.008	F33:A−:B−	J3	9	∼65
p4E2C	rmtB, blaCTX-M-65	SXT	0.015	F33:A−:B−	J4	9	∼65
p1C2J	rmtB, blaCTX-M-65		0.008	F33:A−:B1	J5	9	∼65
pZ425-5	rmtB, blaCTX-M-27		0.004	F33:A−:B−	Smeared	Smeared	∼65

^aRFLP, restriction fragment length polymorphism.

^bPlasmid was obtained from Enterobacter cloacae.

^cPlasmid was obtained from Klebsiella pneumoniae.

^dCHL, chloramphenicol; GEN, gentamicin; NAL, nalidixic acid; STR, streptomycin; TET, tetracycline; SXT, trimethoprim-sulfamethoxazole.

^eFAB, FII:FIA:FIB.

Since most of the rmtB-qepA and rmtB-bla_CTX-M-9G gene combinations were associated with the F2:A−:B− and F33:A−:B− plasmids, respectively (Table 3), these plasmids were subjected to restriction enzyme digestion analysis to clarify whether a specific plasmid had been disseminated among the isolates. Plasmids extracted from the transconjugants or transformants containing only a single plasmid were digested with the endonucleases EcoRI and BamHI (TaKaRa Biotechnology, Dalian, China). Twenty-one F2:A−:B− plasmids carrying both rmtB and qepA were obtained, 18 of which, including one plasmid bearing rmtB, qepA, and bla_CTX-M-14, showed identical plasmid restriction patterns. Interestingly, these patterns are the same as or highly similar to the patterns obtained from F2:A−:B− plasmids carrying both rmtB and qepA in E. coli isolates obtained from pigs, the environment, and farmers in 2002 (Table 3) (7). It is suggested that this F2:A−:B− plasmid is a rather stable plasmid circulating in different members of Enterobacteriaceae and is present in different animal and human reservoirs in China. The F2:A−:B− plasmids are also associated with bla_CTX-M genes in Enterobacteriaceae isolates from China, Hong Kong, South Korea, Vietnam, Italy, Canada, the United Kingdom, and Belgium (12, 14, 20, 24, 26, 30, 34). Further studies are needed to address the mechanisms underlying the worldwide dissemination of these plasmid types.

Twelve F33:A−:B− plasmids carrying both rmtB and bla_CTX-M-9G (including seven carrying bla_CTX-M-65) and one F33:A−:B− plasmid carrying only rmtB showed the same BamHI digestion profiles and highly similar EcoRI digestion profiles (Table 3). bla_CTX-M-65 is one of the dominant CTX-M types in animal isolates obtained in China after 2005 (15, 16, 33) and has also been identified as colocalized with rmtB in pRB1 in an E. coli isolate from a patient in the United States (8). This suggests that the increasing prevalence of CTX-M-65 in E. coli isolates may be due to the dissemination of plasmids carrying both rmtB and bla_CTX-M-65.

In conclusion, the dissemination of rmtB, qepA, and bla_CTX-M genes in Enterobacteriaceae isolates from pets is mediated mainly by the F2:A−:B− and F33:A−:B− plasmids. The coexistence of these resistance determinants in a single plasmid increases the selection by one or more of the antimicrobials used in clinical practice. Therefore, prudent use of antimicrobial agents in pets is urgently needed.

Nucleotide sequence accession numbers.

New replicon sequences described in this work were deposited in the GenBank database with assigned accession numbers GU477621, HQ706665, HQ706666, HQ706667, HQ882837, HQ882838, and HQ882839.