Characterization of Third-Generation Cephalosporin-Resistant Escherichia coli from Bloodstream Infections in Denmark

Abstract

The aim of the study was to investigate the molecular epidemiology of 87 third-generation cephalosporin-resistant Escherichia coli (3GC-R Ec) from bloodstream infections in Denmark from 2009. Sixty-eight of the 87 isolates were extended-spectrum beta-lactamase (ESBL) producers, whereas 17 isolates featured AmpC mutations only (without a coexpressed ESBL enzyme) and 2 isolates were producing CMY-22. The majority (82%) of the ESBL-producing isolates in our study were CTX-M-15 producers and primarily belonged to phylogroup B2 (54.4%) or D (23.5%). Further, one of the two CMY-22-producing isolates belonged to B2, whereas only few of the other AmpCs isolates belonged to B2 and D. Pulsed-field gel electrophoresis revealed that both clonal and nonclonal spread of 3GC-R Ec occurred. ST131 was detected in 50% of ESBL-producing isolates. The remaining ESBL-producing isolates belonged to 17 other sequence types (STs), including several other internationally spreading STs (e.g., ST10, ST69, and ST405). The majority (93%) of the ESBL-producing isolates and one of the CMY-22-producing isolates were multiresistant. In conclusion, 3GC-R in bacteriaemic E. coli in Denmark was mostly due to ESBL production, overexpression of AmpC, and to a lesser extent to plasmid-mediated AmpC. The worldwide disseminated CTX-M-15-ST131 was strongly represented in this collection of Danish, bacteriaemic E. coli isolates.

Introduction

Third-generation cephalosporin-resistant Escherichia coli (3GC-R Ec) is increasing in Europe. During 2002–2009, the proportion of 3GC-R Ec from bloodstream infections has increased from 1.7% to 8.0% in Europe.⁸ A recent European study has shown that bloodstream infections caused by 3GC-R Ec are associated with excess mortality and prolonged hospital stay, posing a considerable burden on patients and healthcare systems.⁵ The extended-spectrum cephalosporin resistance in E. coli can be due to production of extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC (pAmpC), or constitutive overexpression of the chromosomal ampC gene (cAmpC) due to mutations within the promoter/attenuator region.^1,13

The worldwide spread of ESBL-producing E. coli is in part due to the spread of the pandemic clone O25b:-ST131.² Evidently this clone is strongly related to the presence of antimicrobial resistance genes, including bla_CTX-M-15 and other ESBL genes, as well as virulence factors.²⁴

Before 2007 the occurrence of 3GC-R Ec was low among E. coli isolated from bloodstream infections in Danish patients. However, the rate of resistance among invasive E. coli in Denmark has increased from 2.5% in 2006 to 6.2% in 2009.²⁷ In Denmark, the increased frequency of resistance in E. coli blood isolates parallels the increased consumption of broad-spectrum antimicrobial agents including carbapenems.²⁸ Two recent studies from Denmark have characterized ESBL-producing E. coli from 2009; the majority of the isolates in the two studies have been from urinary tract infections, whereas only few isolates were from bloodstream infections.^20,21

The aim of the present study was to investigate 3GC-R Ec isolates obtained from bloodstream infections from four Danish departments of clinical microbiology located in four different regions in Denmark, regarding production of ESBLs, pAmpCs, and cAmpC; pulsed-field gel electrophoresis (PFGE) pattern; affiliation to the multilocus sequence type (MLST) ST131 lineage and other STs, phylogroup types, virulence genes, and coresistance, in order to investigate a possible spread of these isolates in and between the hospitals.

Materials and Methods

Bacterial strains

During January 2009 through December 2009, four Danish departments of clinical microbiology located in Aalborg, North Denmark Region; Odense, Region of Southern Denmark; Naestved (now Slagelse), The Zealand Region; and Hvidovre, The Capital Region of Denmark collected all (n=89) their 3GC-R Ec isolates from bloodstream infections. The 3GC resistance was defined as resistance to cefpodoxime, ceftriaxone, cefotaxime, or ceftazidime. At the clinical department of Aalborg, Odense, and Naestved, Rosco Neosensitabs (Rosco Diagnostica, Taastrup, Denmark) were used and interpreted according to the producer's guideline (NEO-SENSITABS SUSCEPTIBILITY TESTING 19th ed. 2007/2008, Rosco Diagnostica A/S, Taastrup, Denmark), whereas Oxoid disks (Oxoid, Basingstoke, United Kingdom) were used at the clinical department of Hvidovre Hospital, applying breakpoints defined by the Swedish Reference Group for Antibiotics (www.srga.org). E. coli ATCC 25922 was used as control. The four laboratories participated in the external quality assessment by U.K. NEQAS. The isolates were sent to Statens Serum Institut for further testing. Only one isolate per patient was included in the study. Two isolates displayed ESBL-positive phenotypes, but remained ESBL PCR negative and were not included in further analysis. Thus, 87 isolates were included in the study.

Phenotypic screening for ESBL and AmpC

ESBL and/or AmpC phenotypes were confirmed with a combination disk method using Rosco Neosensitabs (Rosco Diagnostica, Taastrup, Denmark) containing cefotaxime (30 μg) and ceftazidime (30 μg) with and without the ESBL inhibitor clavulanic acid (10 μg) and the AmpC inhibitor aminophenylboronic acid (600 μg). A zone of inhibition (ZOI) difference ≥5 mm between an inhibitor combination and the target cephalosporin alone was considered a positive result. Also, ertapenem (10 μg) was included as a screening agent for carbapenemase activity.

Test conditions as described in the European Committee on Antimicrobial Susceptibility Testing (EUCAST) Disc Diffusion Test Methodology (Version 1.0, December 18, 2009) were used for this test.³¹ Briefly, bacterial suspensions equal to an optical density of 0.5 McFarland were inoculated directly on Mueller Hinton Agar plates and incubated for 16–20 hours in ambient air at 35°C after tablet application, before reading of the ZOIs to the nearest mm. E. coli ATCC 25922 was used for quality control.

PCR amplification and sequencing of bla genes or upregulation of AmpC

Based upon the obtained phenotypes, PCR amplification and sequencing was performed to identify the ESBL (TEM, SHV, OXA-2/-10 groups and CTX-M-1/-2/-8 and 9 groups) and plasmid-mediated AmpC (MOX, CMY, LAT, DHA, ACC, MIR-1, ACT-1, and FOX-1 to FOX-5b) enzymes present.^9,11,25 PCR assays were modified to fit the properties of Qiagen Multiplex PCR kit (Qiagen, Hilden, Germany), as per manufacturer's instructions. Positive PCR control strains included Klebsiella pneumoniae KP6T (bla_TEM), K. pneumoniae KP15 (bla_SHV), E. coli J53-2/pM202 (bla_OXA-2), E. coli 14.07 (bla_OXA-10), Salmonella Typhimurium 59.45 (bla_CTX-M-1), Salmonella Virchow 58.67 (bla_CTX-M-2), Salmonella Agona MI.1 (bla_CTX-M-8), Salmonella Virchow 7522438 MOX (bla_CTX-M-9), K. pneumoniae MI.2 (bla_MOX-1), E. coli CF 703-MA (bla_CMY-2), E. coli J53/PMG251 (bla_DHA-1), Salmonella Bareilly 50.32 (bla_ACC-1), E. coli J53/PMG248 (bla_ACT-1), and Salmonella Agona MI.3 (bla_FOX-1).

AmpC-phenotype-positive/plasmid AmpC-negative isolates were investigated for mutations in the AmpC promoter/attenuator region (in comparison with the AmpC nucleotide sequence of E. coli K-12; GenBank, accession No. NC_000913). Purified DNA was sequenced at Macrogen (Seoul, Korea) and analyzed using CLC DNA Workbench 6 (CLC bio, Aarhus, Denmark).

Antimicrobial susceptibility testing

The isolates were susceptibility tested for the following antimicrobial agents: amoxicillin/clavulanic acid, chloramphenicol, ciprofloxacin, colistin, gentamicin, sulfamethoxazole, and trimethroprim, using commercially produced broth dilution panels (Trek Diagnostics, Cleveland, OH) according to the manufacturer's instructions. MIC values were interpreted according to the EUCAST Breakpoint tables for interpretation of MICs and zone diameters (Version 2.0, 2012-01-01). E. coli ATCC 25922 was used for quality control.

PCR for detection of phylogroups

Phylogroup triplex PCR was performed on all isolates as previously described.³ By means of PCR amplification of the group-specific marker genes chuA and yjaA and an anonymous DNA fragment designated TSPE4.C2, the phylogroup could subsequently be determined using the dichotomous decision tree developed by Clermont et al. in the study referred to previously.

Pulsed-field gel electrophoresis

PFGE was performed with XbaI as a restriction enzyme according to Tenover et al.³⁰ Gels (1% agarose) ran for 21 hours in 0.5×TBE with switch times of 2.2–54.2 seconds and voltage of 6.0 Volts/cm. Salmonella Braenderup H9812 was included repeatedly with every run as a normalization standard. The genetic profiles were compared using BioNumerics 6.5 (Applied Maths, Sint-Martens-Latem, Belgium). The levels of similarity between fingerprints were expressed as Dice coefficients, which were calculated by determining the ratio of twice the number of bands shared by two patterns to the total number of bands in both patterns (Optimization: 1%, Tolerance: 1%). Isolates were clustered by using the unweighted pair group method with arithmetic averages (UPGMAs). PFGE clusters were defined with a cutoff value of 80% homology for relatedness.

PCR detection for ST131

An allele-specific PCR of the pabB gene was used to detect isolates belonging to the ST131 clone as described by Clermont et al.²

Multilocus sequence type

From each ESBL-producing non-ST131-related PFGE cluster, one isolate was chosen for MLST. MLST was performed using seven conserved housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) (http://mlst.ucc.ie/mlst/dbs/Ecoli).

Detection of virulence genes by PCR

Virulence factors present in the isolates were investigated using two quadruplex PCR assays previously described by Jakobsen et al.¹⁴ hereby screening for eight ExPEC-related virulence genes: papA, papC, sfaS, and focG (associated with fimbrial adhesion), afa (afimbrial adhesion), kpsM II (group 2 polysaccharide capsule), iutA (uptake of iron), and hlyD (cytolytic protein toxin).

Results

Demographic data

A numeric overweight in isolates from male patients (53 versus 34 female patients) was seen. The average age of patients having the 3GC-R Ec cultured from their bloodstream was slightly above 69 years (range 22–96 years).

Detection of ESBLs, cAmpC, and pAmpCs

In total, 87 isolates were investigated, of which 68 were found to be ESBL positive (Table 1). Twenty-two of these 68 ESBL-positive isolates were also found to have mutations in their AmpC-promoter and/or AmpC-attenuator regions related to overexpression,³² whereas 17 isolates featured these mutations only, without a coexpressed ESBL enzyme (Table 2). In addition to mutations in the AmpC promoter, one isolate was hyperproducing its chromosomal AmpC combined with assumed (based on ertapenem nonsusceptibility and excluded carbapenemase activity) reduced permeability (Table 2). Besides the hyperproducing AmpC isolate, none of the tested isolates were resistant to ertapenem, and were not tested further for the presence of carbapenemases. Finally, two isolates were producing pAmpC, exclusively (Table 2).

Table 1.

Description of the 68 Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates

PFGE	ESBL enzyme	MLST ^a	Clonal complex	Number of isolates	Phylogroup	Virulence profile	Resistance profile (n)	DCM
1	CTX-M-15	ST131	None	12	B2	afa, iutA	AMC, CIP, SUL, TMP (7)	A, H, O, N
							AMC, CIP, GEN, SUL, TMP (1)	O
							AMC, SUL, TMP (2)	A
							AMC, CHL, CIP, SUL, TMP (1)	H
							CIP, SUL, TMP (1)	H
2	CTX-M-15	ST131	None	9	B2	papA, hlyD, iutA, papC	AMC, CIP, GEN, SUL, TMP (7)	H
							CIP, SUL, TMP (1)	H
							AMC, CIP, SUL, TMP (1)	H
3	CTX-M-3	ST131	None	1	B2	kpsM II, iutA	AMC, CIP, TMP (1)	H
3	CTX-M-15	ST131	None	6	B2	kpsM II, iutA	CIP (3)	A, H, O
							AMC, CIP (1)	H
							AMC, CHL, CIP (2)	H
5	CTX-M-15	ST3666	None	3	D	iutA	AMC, CIP, GEN, SUL, TMP (3)	A
6	CTX-M-15	ST167	ST10 Cplx	3	A	iutA	AMC, CIP, SUL, TMP (3)	H
7^b	CTX-M-15	ST88	ST23 Cplx	1 (2)	A	iutA	AMC, CHL, CIP, GEN, SUL, TMP	O
8	CTX-M-15	ST448	ST448 Cplx	2	B1	afa	AMC, CHL, CIP, GEN, SUL, TMP (2)	O
9	CTX-M-15	ST90	ST23 Cplx	2	A	None	AMC, CIP, GEN, SUL, TMP (2)	A
10	CTX-M-15	ST405	ST405 Cplx	2	D	papA, kpsM II, iutA, papC	AMC, CHL, CIP, GEN, SUL, TMP (1)	H
							AMC, CHL, CIP, SUL, TMP (1)	H
11	CTX-M-15	ST131	None	1	B2	papA, kpsM II, hlyD, iutA, papC	AMC, CIP, GEN, SUL, TMP	N
12	CTX-M-15	ST131	None	1	B2	papA, kpsM II, hlyD, iutA, papC	GEN, SUL, TMP	H
13	CTX-M-15	ST131	None	1	B2	papA, kpsM II, iutA, papC	AMC, CIP, SUL, TMP	H
14	CTX-M-15	ST131	None	1	B2	kpsM II, iutA	AMC, CIP, GEN, SUL, TMP	H
15	CTX-M-15	ST131	None	1	B2	iutA	CHL, CIP, SUL, TMP	H
22	CTX-M-15	ST443	ST205 Cplx	1	B1	iutA	CHL, CIP, SUL,	H
23	CTX-M-15	ST10	ST10 Cplx	1	A	None	AMC, CIP, SUL, TMP	O
32	CTX-M-15	ST290	None	1	A	afa	CHL, CIP, SUL, TMP	H
38	CTX-M-15	ST636	None	1	B2	kpsM II, afa, iutA	AMC, SUL, TMP	A
39	CTX-M-15	ST961	None	1	B2	papA, kpsM II, hlyD, papC	AMC, GEN,	A
40	CTX-M-15	ST636	None	1	B2	afa, iutA	AMC, SUL, TMP	A
45	CTX-M-15	ST38	ST38 Clpx	1	D	papA, afa, iutA	AMC, CHL, CIP, GEN, SUL, TMP	O
46	CTX-M-15	ST69	ST69 Clpx	1	D	iutA	SUL, TMP	O
48	CTX-M-15	ST648	None	1	D	papA, kpsM II, iutA, papC	CHL, CIP, SUL, TMP	N
52	CTX-M-15	ST38	ST38 Clpx	1	D	kpsM II	CHL, CIP, SUL, TMP	A
54	CTX-M-15	ST648	None	1	D	kpsM II	AMC, CIP, GEN,	H
24	CTX-M-1	ST10	ST10 Clpx	1	A	afa	SUL,	N
30	CTX-M-1	ST367	ST23 Clpx	1	A	papC	AMC, SUL	H
44	CTX-M-1	ST3929	None	1	D	None	SUL, TMP	O
51	CTX-M-3	ST405	ST405 Clpx	1	D	kpsM II, iutA	AMC, CIP, GEN, SUL, TMP	A
50	CTX-M-9	ST117	None	1	D	iutA, papC	CIP, SUL, TMP	A
26	CTX-M-14	ST88	ST23 Clpx	1	A	papA, iutA, papC	AMC, SUL, TMP	A
47	CTX-M-14	ST38	St38 Clpx	1	D	kpsM II, afa	AMC, CHL, TMP	O
49	CTX-M-14	ST405	St405 Clpx	1	D	None	CIP	A
53	CTX-M-24	ST38	ST38 Clpx	1	D	None	AMC, CHL, SUL, TMP	H
16	CTX-M-27	ST131	None	1	B2	kpsM II, iutA	CIP, SUL, TMP	H
21	TEM-52	ST93	ST138 Clpx	1	Nontypeable	iutA, papC	SUL, TMP	H

From each ESBL-producing non-ST131-related PFGE cluster, one isolate was chosen for MLST.

PFGE type 7 included both a CTX-M-15-producing isolate and a cAmpC isolate, both from DCM O.

AMC, amoxicillin/clavulanic acid; CHL, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; SUL, sulfamethoxazole; TMP, trimethroprim; DCM, department of clinical microbiology; A, Aalborg; H, Hvidovre; N, Naestved; O, Odense.

Table 2.

Description of the 19 AmpC Isolates

Strain ID	PFGE	cAmpC mutation/pAmpC ^a	Phylogroup	Virulence factor profile	Resistance profile	DCM
53-Ec-09	4	6	B2	papA, kpsM II, hlyD, iutA, papC	None	A
75-Ec-09	4	CMY-22	B2	papA, kpsM II, iutA, papC	AMC	H
81-Ec-09	4	2	B2	papA, kpsM II, iutA, papC	None	H
12-Ec-09	7^b	4	A	iutA	None	O
4-Ec-09	17	4	B1		None	O
52-Ec-09	18	5 (+r p)	Nontypeable	papC	AMC	A
96-Ec-09	19	4	Nontypeable		None	H
97-Ec-09	20	1	Nontypeable		None	H
29-Ec-09	25	4	A	afa	None	N
55-Ec-09	27	4	A	papA	None	A
63-Ec-09	28	4	A	papA, iutA, papC	None	H
77-Ec-09	29	4	A	papA, iutA, papC	None	H
88-Ec-09	31	4	A	papA, iutA, papC	None	H
54-Ec-09	34	4	B1	papA, iutA, papC	None	A
56-Ec-09	35	4	B1		None	A
14-Ec-09	36	CMY-22	B2	papA, focG, kpsM II, hlyD, papC	AMC, CHL, SUL	O
21-Ec-09	37	6	B2	focG, kpsM II, afa	None	O
90-Ec-09	42	3	B2	kpsM II, hlyD	None	H
7-Ec-09	43	6	D		None	O

cAmpC mutation: mutations in their AmpC-promoter and/or AmpC-attenuator regions notoriously related to overexpression. pAmpC: plasmid-mediated AmpC.

PFGE type 7 included both a CTX-M-15-producing isolate and a cAmpC isolate both from DCM O.

1, wild-type promoter; 2, wild-type promoter and attenuator; 3, attenuator; 4, alternate, displaced promoter; 5, alternate, displaced promoter and attenuator; 6, outside of functional elements; r p, cAmpC with reduced permeability.

Beta-lactamase gene identification

The majority (56/68; 82%) of the ESBL-positive isolates produced a CTX-M-15 enzyme. In addition to this, CTX-M-1 (n=3), CTX-M-3 (n=2), CTX-M-9 (n=1), CTX-M-14 (n=3), CTX-M-24 (n=1), CTX-M-27 (n=1), and TEM-52 (n=1) were detected (Table 1). The two pAmpC isolates produced CMY-22.

AmpC promoter- and/or -attenuator mutations

All 39 isolates displaying a cAmpC phenotype had their promoter and attenuator regions sequenced. In total, 18 different mutation profiles distributed in six general mutation groups could be observed (data not shown). To assess the impact of the observed mutations the focus below is on the 17 AmpC phenotype/non-ESBL producers found in the study (Tables 2 and 3).

Table 3.

Mutations in the Pre-AmpC Region for 17 cAmpC Escherichia coli Isolates, as Compared with the Sequence of E. coli K12

Strain ID						Promotor										Attenuator							Mutation type ^a	SIR
K12 seq	−88	−82	−73	−55	−42	−32	−28		−18	−14			−1		−16	−19					−37	−57
	C	A	C	T	C	T	G	-	G	T	-	-	C	-	C	C	-	-	-	-	T	C		CTX	CAZ
97-Ec-09	.	G	.	C	.	A	.	-	A	.	-	-	T	-	.	.	-	-	-	-	.	T	1	S	S
81-Ec-09	.	.	T	.	.	A	A	G	.	C	G	T	.	-	T	.	-	-	-	-	C	.	2	I	S
90-Ec-09	.	.	T	.	.	.	A	-	.	.	-	T	.	-	T	.	G	G	G	C	.	.	3	S	S
4-Ec-09	T	G	.	.	T	.	.	-	A	.	-	-	T	-	.	.	-	-	-	-	.	T	4	S	S
12-Ec-09	T	G	.	.	T	.	.	-	A	.	-	-	T	-	.	.	-	-	-	-	.	T		I	I
29-Ec-09	T	G	.	.	T	.	.	-	A	.	-	-	T	-	.	.	-	-	-	-	.	T		I	I
54-Ec-09	T	G	.	.	T	.	.	-	A	.	-	-	T	-	.	.	-	-	-	-	.	T		I	S
55-Ec-09	T	G	.	.	T	.	.	-	A	.	-	-	T	-	.	.	-	-	-	-	.	T		I	I
56-Ec-09	T	G	.	.	T	.	.	-	A	.	-	-	T	-	.	.	-	-	-	-	.	T		I	I
77-Ec-09	T	G	.	.	T	.	.	-	A	.	-	-	T	-	.	.	-	-	-	-	.	T		I	I
88-Ec-09	T	G	.	.	T	.	.	-	A	.	-	-	T	-	.	.	-	-	-	-	.	T		I	I
96-Ec-09	T	G	.	.	T	.	.	-	A	.	-	-	T	-	.	.	-	-	-	-	.	T		R	S
63-Ec-09	T	G	.	.	T	.	.	-	A	.	-	-	T	A	.	.	-	-	-	-	.	T		I	R
52-Ec-09	.	.	.	.	T	.	.	-	A	.	-	-	T	-	.	A	-	-	-	-	.	.	5	R	I
53-Ec-09	.	.	T	.	.	.	A	-	.	G	-	T	.	-	T	.	-	-	-	-	.	.	6	S	S
21-Ec-09	.	.	T	.	.	.	.	-	.	.	G	T	.	-	.	.	-	-	-	-	.	.		S	S
7-Ec-09	.	.	.	.	.	.	.	T	.	.	-	-	.	-	.	.	-	-	-	-	.	.		I	S

Category-defining mutations are in bold. No mutations, as compared to K12, are indicated by dots. Hyphens indicates inserted nucleotides lacking in K12.

1, Mutations in the wild-type promoter; 2, mutations in the wild-type promoter and the attenuator; 3, mutations in the attenuator; 4, mutations that create an alternate, displaced promoter; 5, mutations that create an alternate, displaced promoter and in attenuator; 6, mutations outside of functional elements.

CTX, cefotaxime; CAZ, ceftazidime; S, Susceptible; I, Intermediate; R, Resistant.

As compared with E. coli K-12, one isolate had mutations (including a C to T change in position −42, a G to A change in position −18, and a C to A change in the attenuator region at position 19) creating an alternate, displaced promoter in combination with attenuator mutations, resulting in resistance to cefotaxime and intermediate resistance to ceftazidime. Sequences in another 10 isolates also revealed an alternate, displaced promoter, but lacked attenuator mutations. One of these 10 isolates that were resistant to cefotaxime only, 1 was resistant to ceftazidime only, 1 was intermediate resistant to cefotaxime only, and 6 were intermediate resistant to both, while the remaining isolates were susceptible to both cephalosporins. Seven isolates had mutations in the wild-type promotor and/or the attenuator region, or mutations outside functional elements, which only triggered intermediate resistant or susceptible results for cefotaxime and ceftazidime (Table 3).

Pulsed-field gel electrophoresis

Using a cutoff value of 80% homology for clonal relatedness, PFGE showed a pronounced degree of diversity with a total of 52 different PFGE types among the 87 3GC-R Ec isolates. However, for the ESBL group, three relatively large PFGE clusters were detected, including PFGE-cluster 1 (n=12), PFGE-cluster 2 (n=9), and PFGE-cluster 3 (n=7) (Table 1). The isolates in PFGE-cluster 2 originated from the same hospital in Capital Region of Denmark sending there isolates to Department of Clinical Microbiology Hvidovre, while geographical origin of isolates in the other two clusters (PFGE-clusters 1 and 3) appeared more diverse. Thirteen of the remaining ESBL producers grouped in small clusters: PFGE-cluster 3 (n=3) and PFGE-cluster 6 (n=3); the rest of the ESBL-producing isolates (n=27) were clonally unrelated, that is, <80% similar PFGE profiles. In PFGE-cluster 7, one AmpC isolate clustered with one CTX-M-15 producer (Tables 1 and 2).

Beside PFGE-cluster 7, only one small cluster of three isolates (including one of the CMY-22 producers) (PFGE-cluster 4) was detected among the AmpC-producing isolates (Table 2). The remaining 17 AmpC isolates were clonally unrelated (Table 2).

Identification of ST131

The pabB allele, ST131-specific PCR revealed a presence of this particular sequence type in 34 of the 68 ESBL-producing isolates (50%). They were all CTX-M-15 producers except for two isolates that were producing CTX-M-3 and CTX-M-27, respectively.

The ST131 isolates belonged to the three large PFGE clusters (PFGE-clusters 1, 2, and 3) described earlier. The remaining six ST131 isolates were clonally unrelated by PFGE (Table 1).

Multilocus sequence type

From each ESBL-producing non-ST131-related PFGE cluster (n=27), one isolate was chosen for MLST. The following STs were detected: ST10, ST38, ST69, ST88, ST90, ST93, ST117, ST167, ST290, ST367, ST405, ST443, ST448, ST636, ST648, ST3666, and ST3929 (Table 1).

Phylogenetic typing

Phylogroup B2 was the most common phylogroup (37/68, 54.4%) detected in the ESBL-producing isolates. All the 34 ST131 isolates belonged to phylogroup B2. Further, three of the CTX-M-15 non-ST131 isolates belong to B2 (ST636 [n=2], ST961 [n=1]) (Table 1). While 11 of the ESBL-producing isolates belonged to phylogenetic group A (16.2%), 3 isolates belong to B1 (4.4%), and the remaining 16 isolates belonged to D (23.5%). One isolate was nontypeable.

Among the 17 AmpC-overexpressing isolates, 6 belonged to phylogroup A, 4 belonged to B2, 2 belonged to B1, and a single isolate belonged to group D, while 3 were nontypeable. The two CMY-2-producing isolates belonged to B2 (Table 2).

Virulence genes

All B2 isolates, except one ESBL-producing isolate, were positive for two or more of the tested virulence genes. ESBL-producing isolates belonging to the same PFGE-cluster had identical virulence gene profiles, while one of the cAmpC isolates belonging to PFGE-cluster 4 had a different virulence profile than the other two isolates.

Coresistance

None of the isolates were resistant to colistin. Beside the AmpC-hyperproducing isolate, none of the tested isolates were resistant to ertapenem.

Except for five isolates, all ESBL-producing isolates were resistant to two or more of the tested antimicrobial agents. Different resistant profiles were detected within PFGE-cluster 1, PFGE-cluster 2, and PFGE-cluster 3, whereas all isolates in the smaller clusters with two to three isolates had identical resistant profiles within the PFGE-cluster (Table 2).

None of the 17 cAmpC isolates, except one isolate, were resistant to any of the tested antimicrobial agents. One of the CMY-22-producing isolates was resistant to amoxicillin/clavulanic acid only, whereas the other was resistant to three of the tested antimicrobial agents (Table 2).

Discussion

In the present study the majority of the 3GC-R Ec isolates were from male patients (53 versus 34 female patients). A similar trend has been observed on European level for 3GC-R Ec isolates from bloodstream infections.⁷

In the molecular part of the study of the 87 3GC-R Ec isolates, we found 68 isolates to be ESBL producers, whereas 17 isolates featured AmpC mutations only, without a coexpressed ESBL enzyme, and 2 isolates were producing CMY-22 (pAmpC).

The majority (82%) of the ESBL-producing isolates in our study were CTX-M-15 producers. This is in accordance with the dissemination of CTX-M-15 seen worldwide.²³

Phylogroups B2 and D are associated with highly virulent E. coli isolates,³ and it is therefore very likely to detect a high prevalence of these types among E. coli isolates from bloodstream infections. In our study, the majority of the ESBL-producing isolates belonged to phylogroup B2 (54%) or D (24%). Further, the two CMY-22-producing isolates belonged to B2, whereas only 4 of the 17 cAmpC isolates belonged to B2 and a single isolate belonged to D. All B2 isolates, except one ESBL-producing isolate, were positive for two or more of the tested virulence genes, which with the molecular definition categorizes them as extraintestinal pathogenic E. coli (ExPEC) isolates.¹⁵

ST131 was the most common clonal group among the ESBL-producing isolates, detected in 34 of the 68 ESBL-producing isolates (50%) in our study. A higher fraction of ST131 was found in a study from Israel of the ESBL-producing isolates from bloodstream infections; in that study, 89% of the isolates belonged to ST131.¹⁷ ST131 accounted for 54% of the community-associated ESBL-producing E. coli from centers in the United States.⁶ In two Danish studies, primarily focusing on urinary-tract-infection isolates, 38% and 45% of the isolates belonged to ST131.^20,21 The two other Danish studies and the present study indicate that a large part of the spread of the ESBL-producing isolates in Denmark, as in other parts of the world, is due to the spread of ST131.

A Canadian study showed that the emergence of ST131 among bloodstream isolates started in 2003 and increased in the years 2005–2007.²⁶ We do not know when ST131 was first detected in Denmark, but it seems likely that the emergence of 3GC-R Ec after 2006, in part could be due to the emergence of CTX-M-15-producing E. coli belonging to ST131.

The presence of three large (ST131) and five smaller (non-ST131) PFGE clusters in our study could indicate clonal spread of ESBL-producing E. coli in Danish hospitals, within the same hospital as well as beyond regional borders, or it could be explained by transfer of mobile genetic elements into the same PFGE clusters. The division of ST131 into PFGE clusters has also been observed in a U.K. study by Lau et al.¹⁸ Lau et al. suggested that the multiple clusters within ST131 were a result of DNA rearrangements, mutations, and integration of insertion sequences and other mobile elements. This might also explain the different resistance profiles detected within the PFGE clusters in our study, which might be due to mobile elements or mutations.

Besides ST131, 17 other STs were detected among the 68 ESBL-producing isolates. Several of these STs have been detected internationally. One of the CTX-M-15-producing isolates belonged to ST69 and phylogroup D, known as clonal group A (CgA).¹⁶ This isolate was also resistant to sulfamethoxazole and trimethoprim, which is very common for CgA isolates.¹⁶ Skjøt-Rasmussen et al. found CgA in 15% of E. coli isolates from bloodstream infections with a urinary tract origin collected from January 2003 to May 2005 from four Danish hospitals.²⁹ None of the CgA isolates were ESBL producers in the study by Skjøt-Rasmussen et al., which is in accordance with the low ESBL prevalence in Denmark prior to 2007.²⁹

Isolates belonging to the ST405/phylogroup D lineage are being reported increasingly worldwide. This ST has been associated with multidrug resistance and often with CTX-M-15 production.³⁴ Four of the ESBL-producing isolates (two CTX-M-15, one CTX-M-3, and one CTX-M-14) in our study belonged to ST405. Additionally, four ESBL-producing isolates belonging to ST38 and two isolates belonging to ST648 were detected in our study; these STs have also been detected in a study from the Netherlands.³³ One of the CTX-M-14-producing isolates in our study belonged to ST88. A CTX-M-14-producing ST88 E. coli has been detected in a Danish slaughter pig.¹⁰ Two ESBL-producing isolates in our study belonged to ST10, which have also been detected in veal calves in the Netherlands and in a dog in France, but also from patients in Spain.^4,12,22

In this strain collection, chromosomal AmpC hyperproduction played a less significant role with regards to 3GC resistance as compared to ESBL production, but at least in some isolates, this phenomenon seemed to imply clinical relevance. Other studies have shown that the most important mutation type, with regards to strengthening the AmpC promotor (hereby increasing gene expression), is mutation that creates an alternate, displaced promoter, especially when combined with attenuator mutations (the alternate, displaced promoter having the heaviest impact on expression).³² This type of mutation was seen in 11 (10 of them without attenuator mutations) of the 17 non-ESBL/AmpC phenotypic strains, and they all conferred reduced susceptibility to cefotaxime and/or ceftazidime. Mutations only occurring in the wild-type promotor and/or the attenuator region, or mutations outside functional elements are presumed to play only a minor role in AmpC expression.³² In the remaining isolates, these mutations did not appear to contribute with more than low-level AmpC expression.

The majority (93%) of the ESBL-producing isolates and one of the CMY-22-producing isolates were (besides being resistant to third-generation cephalosporins) resistant to two or more of the tested antimicrobial agents, which make them multidrug resistant according to the international definition proposed by Magiorakos et al.¹⁹

In conclusion, 3GC-R in bacteriaemic E. coli in Denmark was mostly due to ESBL production, overexpression of AmpC, and to a lesser extent to plasmid-mediated AmpC. No carbapenemase producers were detected in this study. CTX-M-15 remains by far the predominant ESBL enzyme type in E. coli in Denmark, representing 82% of all the ESBL-positive E. coli. The worldwide disseminated ST131 was strongly represented in this collection of Danish, bacteriaemic E. coli isolates, as 50% of the ESBL-producing isolates belonged to this clone lineage.

Footnotes

Acknowledgment

This study was supported by the Danish Ministry of Health and Prevention as part of The Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP).

Disclosure Statement

The authors of this article have no commercial associations that might create a conflict of interest in connection with the submitted article.

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