Escherichiacoli with extended-spectrum β-lactamase and plasmid-mediated quinolone resistance genes in great cormorants and mallards in Central Europe

Abstract

Objectives

Faecal Escherichia coli strains were isolated from great cormorants (Phalacrocorax carbo) and mallards (Anas platyrhynchos), which are commonly occurring waterbirds in Europe, and studied for resistance to cephalosporins and fluoroquinolones.

Methods

Cloacal swabs or faeces from great cormorants and mallards in Central Europe were cultivated to isolate Escherichia coli strains with extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes.

Results

Ten ESBL-producing E. coli with the bla_CTX-M-15 or bla_CTX-M-27 gene were isolated from eight great cormorants (1.6%, n = 499). The bla_CTX-M genes were harboured by plasmids of F and I1 incompatibility groups. CTX-M-27-producing isolates were identified as the epidemiologically important B2-O25b-ST131 clone. No ESBL-producing E. coli was isolated from 305 mallards. Eight E. coli isolates with PMQR genes [six aac(6′)-Ib-cr and two qnrS1] were detected in six great cormorants (1.2%). Seventeen strains with qnrS1 were detected in 17 mallards (6%). The PMQR genes were located on plasmids of incompatibility groups F, N or X2. ESBL and PMQR genes were found on conjugative plasmids, enabling the horizontal spread of resistance.

Conclusions

Both great cormorants and mallards can spread epidemiologically important antimicrobial-resistant E. coli isolates to water bodies throughout Europe.

ESBLs, PMQR, Anas, Phalacrocorax, B2-O25b-ST131

Introduction

The emergence of multidrug-resistant bacteria in the natural environment constitutes a serious risk to domestic animal and human health. Wild cormorants and mallards are associated with aquatic environments, to which their search for food is almost exclusively restricted. It can thus be presumed that resistant bacteria colonizing these birds originated in that aquatic environment. The importance of waterbirds, including mallards, in the spread of Escherichia coli resistant to cephalosporins and fluoroquinolones has been recently demonstrated in Poland.¹

In this study, faecal E. coli strains were isolated from cormorants and mallards, which are commonly occurring waterbirds in Europe, and studied for resistance to cephalosporins and fluoroquinolones. The study was focused on extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes and their genetic environments, including possibilities for their horizontal transfer.

Materials and methods

Cloacal swabs or fresh faeces of great cormorants (Phalacrocorax carbo) and mallards (Anas platyrhynchos) were examined. Cloacal swabs were obtained from 49 cormorants shot in the Czech Republic during winter 2006/07 and 2007/08. Moreover, cormorant faeces (n = 300) were collected at a roosting place along the Morava River, Czech Republic, in winter 2007/08. Cormorant faeces (n = 150) were also collected at a roosting place along the Vah River, Slovak Republic, in January 2009. Cloacal swabs from mallards (n = 305) shot at various locations in the Czech Republic were obtained during the autumn hunting season in 2008.

Individual cloacal swabs or faeces samples were enriched in MacConkey broth and then cultivated on MacConkey agar (MCA) with cefotaxime (2 mg/L) and MCA with ciprofloxacin (0.05 mg/L). The E. coli colonies grown on MCA with cefotaxime were examined using the double-disc synergy test for the production of ESBLs.² In the E. coli isolates that were resistant to cefotaxime or ciprofloxacin, susceptibilities to 12 antimicrobial substances were tested using the disc diffusion method according to the CLSI.^1,2E. coli strains were identified using the API 10S test (bioMérieux, Marcy l’Étoile, France). Genes responsible for the ESBL phenotype (bla_TEM, bla_SHV, bla_OXA and bla_CTX-M) were identified by PCR and sequencing.¹ The colonies grown on MCA with ciprofloxacin were tested for PMQR genes [qnrA, qnrB, qnrC, qnrD, qnrS, aac(6′)-Ib-cr and qepA] by PCR, as described previously.¹

All of the other methods used in this study are described elsewhere.³ Briefly, ESBL- and/or PMQR-positive E. coli isolates were typed by XbaI PFGE, and E. coli phylogenetic groups were identified. In isolates belonging to phylogenetic group B2, allele-specific PCR was performed to identify the O25b-ST131 clone of E. coli. Multilocus sequence type (MLST) determination was carried out in isolates positive by allele-specific PCR. Gene amplification and sequencing were performed using primers specified at the E. coli MLST web site (http://mlst.ucc.ie/mlst/mlst/dbs/Ecoli). The insertion sequence ISEcp1 in the upstream region of the bla_CTX-M genes was tested.

The transferability of the bla genes and PMQR genes was tested by conjugation. Plasmid DNA from E. coli was isolated and introduced into competent E. coli DH5α (Invitrogen, USA) by chemical transformation, followed by the selection of transformants on brain heart infusion agar (Oxoid) supplemented with cefotaxime (2 mg/L) or ciprofloxacin (0.05 mg/L). The presence of a relevant bla gene and/or PMQR gene in the transformants was confirmed by PCR. All transformants, transconjugants and their corresponding donors were examined for minimum inhibitory concentrations of ciprofloxacin and nalidixic acid using the agar dilution method.² The size of the plasmids with ESBL or quinolone resistance genes from transformants or transconjugants was designated by S1-PFGE. The restriction fragment length polymorphism (RFLP) of plasmid DNA from transformants/transconjugants was determined. Plasmids were replicon typed as described elsewhere.⁴

Results

Using selective cultivation on MCA with cefotaxime, 10 ESBL-producing E. coli strains were isolated from eight great cormorants (1.6%; n = 499) (Table 1). Their ESBL phenotype was caused by the presence of the bla_CTX-M-27 or bla_CTX-M-15 gene. The insertion sequence ISEcp1 was found upstream from all bla_CTX-M genes. Both CTX-M-27-producing isolates showed the same XbaI-PFGE profile and belonged to the B2-O25b-ST131 lineage (Table 1). All CTX-M-15-producing isolates were multiresistant, and most of them contained aac(6′)-Ib-cr and a class 1 integron, 1.7 kb in length with the gene cassettes dfrA17-aadA5. The bla_CTX-M-15 gene was located on two different plasmid types: IncI1 or IncFIA-FIB (Table 1).

Table 1.

Characterization of ESBL-producing E. coli isolates from great cormorants

Strain no.	AR phenotypea	ESBL bla genes	Additional ARgenes andintegronsb	PFGEprofile	Phylogeneticgroup	Conjugationto E. coli	Conjugationto Salmonella	PlasmidInc group	Plasmidsize (kb)	PlasmidRFLP profile	AR genesand integronson plasmidb
KO 141 B	AMP, CEF	—	—	A	D	−	−	NT	NT	NT
KO 178 B	AMP, CEF, CIP, NAL, STR,SUL, SXT, TET	blaCTX-M-27	strA, sul2, tet(A), Int1	B	B2	−	−	NT	NT	NT
KO 198 B	AMP, CEF, CIP, NAL, STR,SUL, SXT, TET	blaCTX-M-27	strA, sul2, tet(A), Int1	B	B2	−	−	FIA-FIB	120	VII	blaCTX-M-27, strA,tet(A), sul2, Int1
KO 255 B	AMP, CEF, CAZ, CHL, CIP,GEN, NAL, STR, SUL,SXT, TET	blaCTX-M-15	blaTEM-1b, strA, tet(B), cat, Int1	C	D	−	−	NT	NT	NT
KT 58 C	AMP, CEF, CHL, CIP, GEN,NAL, SUL, SXT, TET	blaCTX-M-15	blaOXA-1, aac(6′)-Ib-cr, tet(A),tet(B), cat, sul2, Int1	D	A	+	−	I1	100	V	blaCTX-M-15, sul2,cat, Int1
KT 87c	AMP, CEF, CHL, CIP, NAL,SUL, SXT, TET	blaCTX-M-15	blaOXA-1, aac(6′)-Ib-cr, tet(A),tet(B), cat, sul2, dfr1, Int1	D	A	+	−	FIA-FIB	170	VI	blaCTX-M-15, blaOXA-1,aac(6′)-Ib-cr, tet(A),tet(B), cat, Int1
KT 87 C	AMP, CEF, CHL, CIP,NAL, SUL, SXT, TET	blaCTX-M-15	blaTEM-1b, blaOXA-1, aac(6′)-Ib-cr,tet(A), tet(B), cat, sul2, dfr1,aadA1, sat, Int1	D	A	−	−	NT	NT	NT
KT 121 Bc	AMP, CEF, CHL, CIP, NAL,SUL, SXT, TET	blaCTX-M-15	blaOXA-1, aac(6′)-Ib-cr, tet(A),tet(B), cat, sul2, dfr1, Int1	D	A	−	−	NT	NT	NT
KT 121 C	AMP, CEF, CHL, CIP,NAL, SUL, SXT, TET	blaCTX-M-15	blaOXA-1, aac(6′)-Ib-cr, tet(A),tet(B), cat, sul2, int2, dfr1,aadA2, sat, Int1	D	A	−	−	NT	NT	NT
KT 126 C	AMP, CEF, CHL, CIP NAL,SUL, SXT, TET	blaCTX-M-15	blaOXA-1, aac(6′)-Ib-cr, tet(A),tet(B), cat, sul2, int2, dfr1,aadA2, sat, Int1	D	A	+	−	I1	100	V	blaCTX-M-15, sul2,dfr1, cat, Int1

AR, antibiotic resistance; Inc, incompatibility group; NT, not typeable.

RFLP profile determined by EcoRV and HincII digestion.

^aTwelve substances were tested using the disc diffusion method according to the CLSI: AMC, amoxicillin/clavulanic acid; AMP, ampicillin; CEF, cefalotin; CAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; NAL, nalidixic acid; STR, streptomycin; SUL, sulphonamides; SXT, trimethoprim/sulfamethoxazole; and TET, tetracycline.

^bInt1: sul1, int1, class 1 integron 1.7 kb: dfrA17-aadA5.

^cESBL- and PMQR-positive E. coli strains isolated by selective cultivation with ciprofloxacin (see Table 2).

Using selective cultivation on MCA with ciprofloxacin, four PMQR-positive E. coli strains were isolated from four cormorants (0.8%, n = 499). Two isolates were positive for qnrS1 and another two isolates contained aac(6′)-Ib-cr (Table 2). The qnrS1 gene was located on 50 kb conjugative plasmids of the IncN group with the same RFLP profile in both of the isolates. In the strains carrying aac(6′)-Ib-cr, the genes bla_CTX-M-15 and bla_OXA-1 were detected (Tables 1 and 2).

Table 2.

Characterization of PMQR-positive E. coli from great cormorants and mallards

Origin	Strain no.	AR phenotypea	PMQR genes	Additional AR genesand integronsb	MIC (mg/L)cof NALand CIP		PFGEprofile	Phylogeneticgroup	Conjugationto E. coli	Conjugation toSalmonella	PlasmidIncgroup	Plasmidsize(kb)	PlasmidRFLPprofile	Additional ARgene(s) onplasmid
Cormorants	KT 58 A	STR, TET	qnrS1	strA, tet(A)	128	0.125	f1	A	+	−	N	50	I	strA, tet(A)
	KT 87d	AMP, CHL, CEF, CIP,NAL, SUL, SXT, TET	aac(6′)-Ib-cr	blaCTX-M-15, blaOXA-1, tet(A),tet(B), cat, sul2, dfr1, Int1	>256	>8	D	A	−	−	NT	NT	NT
	KT 121 Bd	AMP, CHL, CEF, CIP,NAL, SUL, SXT, TET	aac(6′)-Ib-cr	blaCTX-M-15, blaOXA-1, tet(A),tet(B), cat, sul2, dfr1, Int1	>256	>8	D	A	−	−	NT	NT	NT
	KT 136 A	STR, TET	qnrS1	strA, tet(A)	128	0.125	f	A	+	+	N	50	I	strA, tet(A)
Mallards	174	AMP, NAL, TET	qnrS1	blaTEM, tet(B)	>256	4	a	A	−	−	NT	NT	NT
	175	TET	qnrS1	—	>256	0.25	b	A	+	−	X2	35	IV	—
	177	TET	qnrS1	tet(B)	128	0.5	c	A	+	−	X2	30	III	—
	178	TET	qnrS1	tet(B)	256	4	c	A	+	+	X2	35	III	—
	188 A	TET	qnrS1	tet(A)	>256	1	d	A	+	+	X2	35	IV	tet(A)
	194	TET	qnrS1	tet(A)	>256	>8	e	B1	+	−	X2	35	IV	tet(A)
	195 A	AMP, CHL, CEF, CIP,NAL, SUL, SXT, TET	qnrS1	blaTEM, tet(B), cat, sul1	>256	>8	NT	B1	+	−	NT	NT	NT
	203 A	TET	qnrS1	tet(A)	>256	>8	e	B1	+	−	X2	40	IV	tet(A)
	204	TET	qnrS1	tet(A)	128	0.25	e	B1	+	−	X2	35	IV	tet(A)
	206	TET	qnrS1	tet(A)	128	0.5	g	A	+	+	X2	35	IV	tet(A)
	209	AMP, TET	qnrS1	blaTEM,tet(A)	128	0.5	h	A	+	+	N	45	II	blaTEM, tet(A)
	212	TET	qnrS1	tet(A)	>256	0.5	i	A	+	−	X2	35	IV	tet(A)
	220	TET	qnrS1	tet(A)	128	0.5	j	A	+	+	X2	35	IV	tet(A)
	226 B	TET	qnrS1	tet(A)	>256	0.5	k	A	+	+	X2	35	IV	tet(A)
	231	AMP	qnrS1	blaTEM	>256	0.5	l	A	+	+	N	40	IIa	blaTEM
	234	TET	qnrS1	tet(A)	64	0.25	m	A	+	+	X2	40	IV	tet(A)
	242	TET	qnrS1	tet(A)	128	0.25	j	A	+	−	X2	40	IV	tet(A)

AR, antibiotic resistance; Inc, incompatibility group; NT, not typeable.

RFLP profile determined by EcoRV and HincII digestion.

^aTwelve substances were tested using the disc diffusion method according to the CLSI: AMC, amoxicillin/clavulanic acid; AMP, ampicillin; CEF, cefalotin; CAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; NAL, nalidixic acid; STR, streptomycin; SUL, sulphonamides; SXT, trimethoprim/sulfamethoxazole; and TET, tetracycline.

^bInt1: sul1, int1, class 1 integron 1.7 kb: dfrA17-aadA5.

^cMIC of nalidixic acid and ciprofloxacin for donor strains.

^dE. coli strains positive for PMQR and ESBL genes (see Table 1).

No ESBL-producing E. coli isolate was detected in mallards. Using selective cultivation with ciprofloxacin, 17 qnrS1-positive E. coli isolates were detected in 17 mallards (6%, n = 305) (Table 2). The qnrS1 gene was transferred by conjugation to E. coli and Salmonella. The qnrS1 gene was located on conjugative plasmids of incompatibility groups X2 or N that ranged in size from 30 to 45 kb (Table 2).

As determined by the χ² test, the prevalence of ESBL-positive E. coli isolates in cormorants was statistically higher than in mallards and, in contrast, the prevalence of PMQR-positive strains in mallards was statistically higher than in cormorants.

Discussion

CTX-M enzymes are currently the most prevalent ESBLs in the world and they are identified mainly in E. coli.⁵ We report here E. coli isolates with ESBL genes in cormorants for the first time. Great cormorants and mallards are common waterbirds in Central Europe, and thus the detection of CTX-M-type ESBL and PMQR genes in these wild birds may indicate a rapid spread of these resistance genes within the environment. As these waterbirds are almost exclusively associated with the water environment, we suppose that they were colonized with resistant E. coli strains from this environment; they can then become important reservoirs and potential vectors of the bacteria in the water environment.

In the present study, the bla_CTX-M-15 gene was harboured on conjugative plasmids of the incompatibility groups IncF and IncI1. The association between the genes bla_CTX-M-15, bla_OXA-1 and aac(6′)-Ib-cr and the IncF plasmid was recently described in clinical and food animal E. coli isolates.⁶ Plasmids belonging to the IncI1 group carrying bla_CTX-M-15 have also recently been described in E. coli isolates.⁷

In our study, the bla_CTX-M-27 gene was found in two epidemiologically related E. coli isolates from cormorants. To our knowledge, this is the first report of CTX-M-27-producing E. coli in wild animals. Moreover, these CTX-M-27-positive isolates belonging to the B2 phylogenetic group were identified as the O25b-ST131 clone, which has high virulence potential all over the world and represents a major public health problem. This is the first detection of a CTX-M-27 ESBL type in an O25b-ST131 isolate in Europe. To date, the only finding of a CTX-M-27-producing E. coli clone B2-O25-ST131 has been reported in China.⁸

In this study, E. coli isolates with qnrS1 were detected at a prevalence of 6% in mallards. Recently, four E. coli isolates with qnrS genes were found in mallards from the Polish coast of the Baltic Sea.¹ In all E. coli strains from Central European mallards, the qnrS1 gene was located on transferable plasmids of incompatibility groups X2 or N. The IncX2 plasmid is not common, and the association between this plasmid and qnrS has been described only very recently.^1,9 In four E. coli isolates, qnrS1 was found to be located on conjugative plasmids of the IncN group. The association of qnrS and IncN plasmids has been recently reported.^1,10

Selective pressure or sources of colonization probably differ in great cormorants and mallards, as shown by the different prevalences of ESBL-producing and PMQR-positive E. coli isolates in great cormorants and mallards, respectively. As common waterbirds, both great cormorants and mallards can spread epidemiologically important antimicrobial-resistant E. coli, including the pandemic O25b-ST131 clone.

Funding

This study was funded by the Czech Science Foundation (grant no. P502/10/P083). This study was also supported by the project ‘CEITEC—Central European Institute of Technology’ (CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund.

Transparency declarations

None to declare.