Molecular Epidemiological Analysis of Escherichia coli Sequence Type ST131 (O25:H4) and bla_CTX-M-15 among Extended-Spectrum-β-Lactamase-Producing E. coli from the United States, 2000 to 2009

Abstract

Escherichia coli sequence type ST131 (from phylogenetic group B2), often carrying the extended-spectrum-β-lactamase (ESBL) gene bla_CTX-M-15, is an emerging globally disseminated pathogen that has received comparatively little attention in the United States. Accordingly, a convenience sample of 351 ESBL-producing E. coli isolates from 15 U.S. centers (collected in 2000 to 2009) underwent PCR-based phylotyping and detection of ST131 and bla_CTX-M-15. A total of 200 isolates, comprising 4 groups of 50 isolates each that were (i) bla_CTX-M-15 negative non-ST131, (ii) bla_CTX-M-15 positive non-ST131, (iii) bla_CTX-M-15 negative ST131, or (iv) bla_CTX-M-15 positive ST131, also underwent virulence genotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). Overall, 201 (57%) isolates exhibited bla_CTX-M-15, whereas 165 (47%) were ST131. ST131 accounted for 56% of bla_CTX-M-15-positive- versus 35% of bla_CTX-M-15-negative isolates (P < 0.001). Whereas ST131 accounted for 94% of the 175 total group B2 isolates, non-ST131 isolates were phylogenetically distributed by bla_CTX-M-15 status, with groups A (bla_CTX-M-15-positive isolates) and D (bla_CTX-M-15-negative isolates) predominating. Both bla_CTX-M-15 and ST131 occurred at all participating centers, were recovered from children and adults, increased significantly in prevalence post-2003, and were associated with molecularly inferred virulence. Compared with non-ST131 isolates, ST131 isolates had higher virulence scores, distinctive virulence profiles, and more-homogeneous PFGE profiles. bla_CTX-M-15 was associated with extensive antimicrobial resistance and ST131 with fluoroquinolone resistance. Thus, E. coli ST131 and bla_CTX-M-15 are emergent, widely distributed, and predominant among ESBL-positive E. coli strains in the United States, among children and adults alike. Enhanced virulence and antimicrobial resistance have likely promoted the epidemiological success of these emerging public health threats.

INTRODUCTION

Extraintestinal Escherichia coli infections, including urinary tract infections, sepsis, and neonatal meningitis, are important causes of morbidity, mortality, and increased health care costs (26). Their management is increasingly challenging due to the rising prevalence of resistance to first-line antimicrobial agents, including fluoroquinolones and extended-spectrum cephalosporins (14, 23). Contributing significantly to this increase is a recently emerged E. coli lineage designated sequence type ST131, which is characterized as belonging to serotype O25:H4 and is associated with fluoroquinolone resistance and CTX-M-15, an IncFII-plasmid-mediated extended-spectrum β-lactamase (ESBL) (21, 25).

The initial reports in 2008 of E. coli ST131 as an important new pathogen involved isolates from diverse non-U.S. international locales (3, 5, 18). Subsequently, case reports and small series documented the presence of ST131 also in the United States, with the ESBL-positive ST131 isolates usually producing CTX-M-15 (6, 7, 9, 11, 17, 20, 27, 29, 30). The only published large-scale survey to date for ST131 in the United States appeared in 2010 and was based on clinical isolates from 2007 from two national surveillance programs (8). There, ST131 accounted for approximately two-thirds of all ESBL-positive or fluoroquinolone-resistant E. coli study isolates, with 73% of ESBL-positive ST131 isolates exhibiting CTX-M-15 (8).

Limitations of that study included the single study year, restrictions with respect to the participating sites and selection criteria of the surveillance systems, and inclusion of only 59 total ESBL-positive isolates (8). Here we assessed the reproducibility of that survey's findings and explored new associations, using a 10-year study period and a much larger population that included isolates from both children and adults.

MATERIALS AND METHODS

Strains.

The 351 ESBL-positive E. coli study isolates, including some previously published isolates (16, 17, 27, 29), were obtained as a series of convenience samples from colleagues at 15 research or clinical laboratories across the United States (Table 1). Each center was invited to submit all available E. coli clinical isolates that met the study criteria, including (i) isolation from 2000 to 2009 and (ii) confirmed ESBL production according to Clinical and Laboratory Standards Institute (CLSI) criteria (4). Upon receipt in the study laboratory, all isolates were screened for bla_CTX-M-15 by PCR (as described below). All bla_CTX-M-15 PCR-negative isolates were further screened by disk diffusion for extended-spectrum-cephalosporin susceptibility (as described below). All extended-spectrum-cephalosporin-susceptible isolates so identified were further screened for ESBL production, using cefotaxime and ceftazidime disks with or without clavulanate (4). For study inclusion, isolates were required to be bla_CTX-M-15 positive or, if bla_CTX-M-15 negative, to be either extended-spectrum cephalosporin resistant or (if retested) to exhibit an ESBL phenotype.

Table 1.

Sources and ST131 and bla_CTX-M-15 status of 351 extended-spectrum-β-lactamase-producing Escherichia coli isolates collected in the United States in 2000 to 2009

City or center source (reference, if published)	Yr of isolation	Patient population	Total no. of isolates	No. (%) of isolates in indicated categorya,b						No. of ST131 isolates/total no. of isolates (%)a
				Trait		Subseta,b
				M15+	ST131+	ST131− M15−	ST131− M15+	ST131+ M15−	ST131+ M15+	M15−	M15+
Miami, FL	2009	Adultsc	23	19 (83)	7 (30)	1 (4)	15 (65)	3 (13)	4 (17)	3/4 (75)	4/19 (21)
Chicago, IL	2007–2008	Adults	8	6 (75)	5 (63)	1 (13)	2 (25)	1 (13)	4 (50)	1/2 (50)	4/6 (67)
Evanston, IL	2007	Adults	27	16 (59)	17 (63)	4 (15)	6 (22)	7 (26)	10 (37)	7/11 (64)	10/16 (63)
Portland, ME	2008	Adult	1	1 (100)	1 (100)	0 (0)	0 (0)	0 (0)	1 (100)	(none)	1/1 (100)
Minneapolis, MN (Hennepin County Medical Center)	2008–2009	Adults	31	17 (55)	17 (55)	9 (29)	5 (16)	5 (16)	12 (39)	5/14 (36)	12/17 (71)
Minneapolis, MN (Children's Hospital)	2002–2009	Children	33	12 (36)	12 (36)	16 (48)	5 (15)	5 (15)	7 (21)	5/21 (24)	7/12 (58)
St. Paul, MN	2007–2008	Adults	20	12 (60)	7 (35)	5 (25)	8 (40)	3 (15)	4 (20)	3/8 (38)	4/12 (33)
Winston-Salem, NC	2008–2009	Adults	11	8 (73)	8 (73)	3 (27)	0 (0)	0 (0)	8 (73)	0 (0)	8/8 (100)
Omaha, NE	2005	Adults	4	1 (25)	2 (50)	2 (50)	0 (0)	1 (25)	1 (25)	1/3 (33)	1/1 (100)
Philadelphia, PA (17)	2007	Adults	31	14 (45)	15 (48)	10 (32)	6 (19)	7 (23)	8 (26)	7/17 (41)	8/14 (57)
Pittsburgh, PA (27)	2006–2007	Adults	17	7 (41)	10 (59)	5 (29)	2 (12)	5 (29)	5 (29)	5/10 (50)	5/7 (71)
New York City (Queens), NY (29)	2004–2008	Adult	63	37 (59)	38 (60)	15 (24)	10 (16)	11 (17)	27 (43)	11/26 (42)	27/37 (73)
Houston, TX (30)	2004–2009	Adults	38	30 (79)	10 (26)	7 (18)	21 (53)	1 (3)	9 (24)	1/8 (13)	9/30 (30)
San Antonio, TX (16)	2000–2006	Adults	20	11 (55)	7 (35)	7 (35)	6 (30)	2 (10)	5 (25)	2/9 (22)	5/11 (45)
Seattle, WA	2004–2007	Childrend	24	10 (42)	9 (38)	13 (54)	2 (8)	1 (4)	8 (33)	1/14 (7)	8/10 (80)
Total	2000–2009	Mixed	351	201 (57)	165 (47)	97 (28)	89 (25)	51 (15)	114 (33)	51/148 (34)	114/203 (56)

^aM15⁺, positive for bla_CTX-M-15; M15⁻, negative for bla_CTX-M-15.

^bST131⁺, ST131 sequence type; ST131⁻, non-ST131 sequence type.

^cIncludes 1 pediatric isolate.

^dIncludes 1 adult isolate.

Detection of bla_CTX-M-15.

The PCR screen for bla_CTX-M-15 involved a combination of published and newly devised primers. After an initial screen using primers that detect bla_CTX-M-15 plus related bla_CTX-M variants (15), PCR-positive isolates were retested using a novel extended “CTX-M-15-specific” forward primer (15extF [ATAAAACCGGCAGCGGTGG]), which hybridizes with bla_CTX-M-15 and, per the data shown at http://www.lahey.org/Studies/other.asp#table1, with several other group 1 bla_CTX-M variants (including bla_{CTX-M-28, -29, 3-3, -53, -82} but not bla_CTX-M-1, bla_CTX-M-3, or several other bla_CTX-M-3-like variants detected by the bla_CTX-M-15 screening primers). They also were retested using a novel extended “CTX-M-3-specific” forward primer (3extF [ATAAAACCGGCAGCGGTGA]), which hybridizes with bla_CTX-M-3 and a different set of group 1 bla_CTX-M variants (including bla_{CTX-M-1, -10, -11, -12, -22, -23, -30, -32, -34, -36, -37, -42, -52}). Isolates reacting with the extended bla_CTX-M-15 primer but not the extended bla_CTX-M-3 primer were further screened using a novel bla_CTX-M-28-specific forward primer (28for [GGTTAAAAAATCACTGCGT]), in combination with CTX-M group 1 reverse primer M13L (15), since bla_CTX-M-28 is the most frequent of the potential cross-reacting bla_CTX-M variants.

Conditions for the novel PCRs were largely as described elsewhere (13). Reaction mixtures included 1× buffer, 4 mM MgCl₂, 0.8 mM deoxynucleoside triphosphate (dNTP) mix, 1.25 U of AmpliTaq Gold, 0.6 μM specific primers, 2.0 μl of boiled lysate DNA, and H₂O to reach a final volume of 25 μl. Cycling was performed as follows: 95°C activation for 10 min; 25 cycles of 30 s of denaturation at 94°C and then 3 min of annealing and extension at 76°C (for bla_CTX-M-15 and bla_CTX-M-3 PCR [amplicon, 483 bp]) or at 68°C (for bla_CTX-M-28 PCR [amplicon, 863 bp]); 10′ extension at 72°C; and then a hold at 4°C. Isolates that gave positive test results with the initial and extended bla_CTX-M-15 primers but negative test results with the bla_CTX-M-3 and bla_CTX-M-28 primers were regarded as bla_CTX-M-15 positive.

Validation of this tiered PCR approach using 257 reference isolates containing sequenced bla_CTX-M variants (181 reportedly bla_CTX-M-15, 76 reportedly non-bla_CTX-M-15) yielded only 9 discrepancies, all but three of which were resolved in favor of the PCR results by repeat PCR and sequencing. The persisting discrepancies involved 3 bla_CTX-M-53-positive isolates that were falsely identified by PCR as containing bla_CTX-M-15, consistent with the predicted cross-reactivity of bla_CTX-M-15 primers with the (rarely encountered) bla_CTX-M-53 variant. This yielded an estimated overall accuracy rate of 98.8% (95% confidence interval, 96.6% to 99.8%) for detection of bla_CTX-M-15 by tiered PCR.

Phylogenetic group and ST131 status.

The major E. coli phylogenetic groups (A, B1, B2, and D) were determined by triplex PCR (1). ST131 isolates were identified by PCR-based detection of ST131-specific single-nucleotide polymorphisms (SNPs) in mdh and gyrB (10). Seventeen randomly selected presumptive ST131 isolates underwent confirmatory 7-locus multilocus sequence typing (MLST) based on partial sequences for adk, fumC, gyrB, icd, mdh, purA, and recA (http://mlst.ucc.ie/mlst/dbs/Ecoli); all were confirmed as ST131. All isolates were screened by PCR for ST131-associated traits, including the F10 papA allele (P-antigen-recognizing fimbria [P fimbria] structural subunit variant) and the O25b rfb (lipopolysaccharide synthesis) variant (2, 10).

Antimicrobial susceptibility.

A total of 200 isolates, comprising 4 groups of 50 randomly selected isolates each that were (i) bla_CTX-M-15 negative non-ST131; (ii) bla_CTX-M-15 positive non-ST131; (iii) bla_CTX-M-15 negative ST131; or (iv) bla_CTX-M-15-positive ST131 underwent additional testing. Antimicrobial susceptibility to 21 agents was determined by disk diffusion using Clinical and Laboratory Standards Institute (CLSI)-specified procedures, control strains, and (2011) interpretive criteria. Agents tested included amikacin (AMK), ampicillin (AMP), amoxicillin-clavulanate (AML), aztreonam (ATM), cefepime (FEP), ceftazidime (CAZ), ceftriaxone (CRO), cephalothin (KF), chloramphenicol (C), ciprofloxacin (CIP), gentamicin (CN), imipenem (IMP), nalidixic acid (NA), nitrofurantoin (F), piperacillin (PIP), piperacillin-tazobactam (TZP), streptomycin (S), sulfonamides (SF), tetracycline (TE), trimethoprim (W), and trimethoprim-sulfamethoxazole (SXT). Intermediate results were analyzed as representing resistance. The resistance score represented the number of agents to which an isolate was resistant, excluding SXT (due to redundancy with SF and W).

Extended virulence genotypes.

The 200 isolates were tested for the presence of 50 extraintestinal pathogenic E. coli (ExPEC)-associated virulence genes and housekeeping gene uidA (β-glucuronidase) by multiplex PCR (10). Isolates were defined as ExPEC if positive for ≥2 of papA and/or papC (P fimbria major subunit and assembly), sfa/focDE (S and F1C fimbriae), afa/draBC (Dr-binding adhesins), kpsM II (group 2 capsule), and iutA (aerobactin receptor) (12). The virulence score represented the number of virulence genes detected, adjusted for multiple detection of the pap, sfa, foc, and kpsM II operons.

PFGE analysis.

The 200 isolates underwent pulsed-field gel electrophoresis (PFGE) analysis of XbaI-restricted total DNA per the PulseNet protocol (24). By using BioNumerics (Applied Maths) and band-based Dice similarity coefficients, pulsotype designations were assigned at the ≥94% profile similarity level, corresponding to an approximately 3-band difference (28).

Statistical methods. Comparisons of proportions were tested using Fisher's exact test (two-tailed) or a chi-square test if unpaired and McNemar's test if paired. Comparisons of virulence scores were tested using the Mann-Whitney U test (two-tailed). To simplify the virulence genotype and antimicrobial susceptibility data for comparisons among the four subgroups as defined by combined CTX-M-15 status and ST131 status, principal coordinate analysis (PCoA) was performed using GENALEX 6 software (19). This multidimensional scaling method captures within the first 2 axes most of the total variance contained in a multivariable, multispecimen data set. Analysis of molecular variance (AMOVA) was used to determine the percentage of diversity within populations versus among populations (19).

RESULTS

Origins of isolates.

The 351 ESBL-producing E. coli study isolates were from 15 centers distributed broadly across the United States (Table 1). Centers contributed a median of 23 isolates each (range, 1 to 63 isolates). Years of isolation ranged from 2000 to 2009 (median, 2007). Source patients included both adults (n = 295 [84%]) and children (n = 57 [16%]). Additional clinical data, including specimen type, were unavailable.

CTX-M-15 and ST131 status.

According to tiered PCR-based testing, 201 (57%) of the 351 study isolates contained CTX-M-15 (median per center, 59%; range, 25% to 100%), whereas 165 (47%) represented ST131 (median per center, 50%; range, 26% to 100%) (Table 1). Overall, ST131 was statistically significantly associated with CTX-M-15, accounting for 56% of CTX-M-15-positive isolates versus 34% of CTX-M-15-negative isolates (P < 0.001). This trend was evident at 12 of the 15 individual centers, including both children's hospitals (Table 1).

The 4 possible combinations of ST131 status and CTX-M-15 status defined 4 subgroups (Table 1). Of these, the dual-positive subgroup (i.e., ST131⁺ CTX-M-15⁺) was the most prevalent (33%), followed sequentially by the ST131⁻ CTX-M-15⁻ subgroup (28%), the ST131⁻ CTX-M-15⁺ subgroup (25%), and the ST131⁺ CTX-M-15⁻ subgroup (15%) (Table 1). All but three centers contributed one or more representatives of each of these 4 subgroups, and the 3 centers that did not do so provided only 1 to 11 isolates each.

CTX-M-15 and ST131 versus phylogenetic group, F10 papA allele, and O25b rfb variant.

In the total population, group B2 was the dominant phylogenetic group, accounting for 50% of isolates, followed sequentially in prevalence by groups D (25%), A (18%), and B1 (7%) (Table 2). Whereas by definition the ST131 isolates were all from group B2, regardless of CTX-M-15 status, the non-ST131 isolates were least likely to be from group B2 (versus other groups), and their phylogenetic group distribution varied significantly with CTX-M-15 status. That is, the CTX-M-15-negative non-ST131 isolates were predominantly from group D, followed by groups B1, A, and B2. In contrast, the CTX-M-15-positive non-ST131 isolates were predominantly from group A, followed by groups D and B1, with none from group B2.

Table 2.

Characteristics of 351 extended-spectrum-β-lactamase-producing Escherichia coli clinical isolates from the United States, 2000 to 2009

Characteristica	No. (%) of isolatesb					P valuec
	Total (n = 351)	Subset
		ST131− M15− (group 1; n = 98)	ST131− M15+ (group 2; n = 89)	ST131+ M15− (group 3; n = 52)	ST131+ M15+ (group 4; n = 113)	Group 1 vs 2	Group 3 vs 4	Group 1 vs 3	Group 2 vs 4
Group A	63 (18)	17 (17)	46 (52)	0 (0)	0 (0)	< 0.001		0.001	< 0.001
Group B1	24 (7)	20 (20)	4 (5)	0 (0)	0 (0)	0.002		< 0.001	0.035
Group B2	175 (50)	10 (10)	0 (0)	52 (100)	113 (100)	0.002		< 0.001	< 0.001
Group D	89 (25)	51 (52)	38 (43)	0 (0)	0 (0)			< 0.001	< 0.001
F10 papA allele	168 (48)	9 (9)	8 (9)	42 (81)	109 (97)		0.002	< 0.001	< 0.001
O25b rfb allele	159 (45)	0 (0)	0 (0)	48 (92)	110 (97)			< 0.001	< 0.001
Post-2003 origin	338 (96)	89 (91)	85 (97)	51 (98)	113 (100)			0.10	0.08

^aGroup, major E. coli phylogenetic group; F10 papA allele, P fimbria structural subunit variant; O25b rfb allele, O25 lipopolysaccharide synthesis.

^bM15⁺, positive for bla_CTX-M-15; M15⁻, negative for bla_CTX-M-15; ST131⁺, ST131 sequence type; ST131⁻, non-ST131 sequence type.

^cP values (by Fisher's exact test; two-tailed) are shown where P ≤ 0.10. For all variables, an initial 4-group comparison yielded P ≤ 0.003.

The F10 papA allele and O25 rfb variant were significantly more prevalent among ST131 isolates than non-ST131 isolates, regardless of CTX-M-15 status (Table 1). Additionally, for the ST131 isolates, the F10 papA allele was significantly more prevalent among CTX-M-15-positive than CTX-M-15-negative isolates, although the absolute difference was modest (81% versus 97%) (Table 1).

CTX-M-15 and ST131 versus year of isolation.

When analyzed as a continuous variable, the year of isolation was borderline significantly more recent for CTX-M-15-positive isolates than CTX-M-15-negative isolates (P = 0.099) and for ST131 isolates than non-ST131 isolates (P = 0.053). The distribution of isolation years by CTX-M-15 and ST131 status suggested that, for both variables, 2003 to 2004 would be the optimal breakpoint for dichotomous stratification (not shown). With the years categorized using this breakpoint, CTX-M-15 and ST131 were both significantly more prevalent among isolates from 2004 to 2009 than among those from 2000 to 2003 (for CTX-M-15, 77% versus 23% [P = 0.019]; for ST131, 49% versus 8% [P = 0.004]), consistent with the emergence of both traits over the decade.

Among the four subgroups defined by combined CTX-M-15 status and ST131 status, the proportion of isolates from 2004 to 2009 (versus isolates from 2000 to 2003) increased progressively across subgroups, from a low of 91% (CTX-M-15-negative, non-ST131 isolates) to a high of 100% (CTX-M-15-positive, ST131 isolates) (P = 0.003 for the 4-group comparison) (Table 2). In pairwise comparisons among the 4 subgroups, ST131 isolates were borderline significantly more likely than non-ST131 isolates to be from 2004 to 2009, regardless of CTX-M-15 status (Table 2).

Extended virulence genotypes.

Genotypes for 51 ExPEC-associated virulence markers were determined for 50 randomly selected representatives per subgroup for the 4 subgroups as defined by CTX-M-15 status and ST131 status (total n = 200). The selected isolates exhibited the same phylogenetic group distribution as their respective source subgroups, evidence that they were a representative subset (Table 3). All but 8 of the virulence markers sought were detected in ≥1 isolate each, with 18 (42%) of the detected markers exhibiting a significant between-subgroup prevalence difference in relation to CTX-M-15 status, ST131 status, or both (Table 3).

Table 3.

Molecular characteristics of 200 selected extended-spectrum-β-lactamase-producing Escherichia coli isolates (collected in 2000 to 2009) in relation to ST131 and bla_CTX-M-15 status

Trait categorya,b	Specific traita	No. (%) of isolates				P valuec
		ST131− M15− (group 1; n = 50)	ST131− M15+ (group 2; n = 50)	ST131+ M15− (group 3; n = 50)	ST131+ M15+ (group 4; n = 50)	Group 1 vs 2	Group 3 vs 4	Group 1 vs 3	Group 2 vs 4
Phy. group	Group A	10 (20)	28 (56)	0 (0)	0 (0)	<0.001		<0.001	<0.001
	Group B1	7 (14)	1 (2)	0 (0)	0 (0)			0.01
	Group B2	5 (10)	0 (0)	50 (100)	50 (100)			<0.001	<0.001
	Group D	28 (56)	21 (42)	0 (0)	0 (0)			<0.001	<0.001
Adhesins	F10 papA	4 (8)	5 (10)	40 (80)	50 (100)		0.001	<0.001	<0.001
	afa/draBC	3 (6)	2 (4)	2 (4)	13 (26)		0.03		0.004
	iha	8 (16)	3 (6)	39 (78)	48 (96)		0.02	<0.001	<0.001
	fimH	42 (84)	30 (60)	50 (100)	50 (100)	0.01		0.006	<0.001
	hra	10 (20)	6 (12)	1 (2)	4 (8)			0.008
Toxins	sat	6 (12)	3 (6)	39 (78)	48 (96)		0.02	<0.001	<0.001
	vat	6 (12)	0 (0)	1 (2)	0 (0)	0.03
	astA	9 (18)	5 (10)	0 (0)	0 (0)			0.03
Siderophores	fyuA	30 (60)	31 (62)	49 (98)	50 (100)			<0.001	<0.001
	iutA	21 (42)	39 (78)	43 (86)	49 (98)	<0.001		<0.001	<0.001
Capsule	kpsM II	18 (36)	11 (22)	35 (70)	45 (90)		0.02	0.001	<0.001
	K2	3 (6)	4 (8)	5 (10)	17 (34)		0.007		0.003
	K5	3 (6)	0 (0)	20 (40)	18 (36)			<0.001	<0.001
	kpsMT III	3 (6)	9 (18)	0 (0)	0 (0)				0.003
Miscellaneous	usp	7 (14)	1 (2)	49 (98)	50 (100)			<0.001	<0.001
	ompT	15 (30)	7 (14)	48 (96)	50 (100)			<0.001	<0.001
	traT	34 (68)	42 (84)	44 (88)	46 (92)			0.03
	malX	17 (34)	20 (40)	48 (96)	50 (100)			<0.001	<0.001
ExPEC	n.a.d	15 (30)	7 (14)	33 (66)	47 (94)		0.001	0.001	<0.001
PFGE statuse	≥2 per type	9 (19)f	22 (44)	27 (54)	41 (82)	0.009	0.005	<0.001	<0.001
	≥5 per type	0 (0)	0 (0)	18 (36)	33 (66)			<0.001	<0.001

^aTraits shown are those that yielded P < 0.05 in an initial four-way comparison (not shown) plus at least one pairwise comparison (as shown). These included afa/draBC (Dr-binding adhesins), astA (enteroaggregative E. coli heat-stable toxin), the F10 papA allele (P fimbria structural subunit variant), fimH (type 1 fimbria adhesin), fyuA (yersiniabactin system), hra (heat-resistant agglutinin), iha (adhesin siderophore), iutA (aerobactin system), K2 (group 2 capsule variant), K5 (group 2 capsule variant), kpsMII (group 2 capsule), kpsMTIII (group 3 capsule), malX (fitness island marker), ompT (outer membrane protease), sat (secreted autotransporter toxin), usp (uropathogenic specific protein), and vat (vacuolating toxin). Traits detected in ≥1 isolate but without significant by-group prevalence differences (definition: overall prevalence) included afaE8 (afimbrial adhesin: 1.5%), bmaE (M fimbriae: 1.5%), cdtB (cytolethal distending toxin B: 0.5%), clbB and clbN (polyketide synthesis: 1% and 2%), clpG (mannose-resistant adhesin: 0.5%), cnf1 (cytotoxic necrotizing factor: 2%), cvaC (microcin V: 1%), H7 fliC allele (flagellin: 0.5%), hlyD (alpha hemolysin: 3.5%), hlyF (hemolysin variant: 4%), ibeA (invasion of brain endothelium: 2%), ireA (siderophore receptor: 1.5%), iroN (salmochelin receptor: 3%), iss (increased serum survival: 3%), K1 (capsule variant: 3%), papAH (P fimbria major subunit: 4.5%), papC (P fimbria assembly: 7.0%), papEFG (P fimbria tip pilins: 5.5%), papG alleles II and III (P adhesin variants: 4.5% and 1%), pic (autotransporter protease: 0.5%), rfc (O4 lipopolysaccharide synthesis: 1%), traT (serum resistance associated: 82.5%), and tsh (temperature-sensitive hemagglutinin: 2%). Traits sought but not detected included F17 (mannose-resistant adhesin), focG (F1C adhesin), gafD (G fimbriae), K15 (capsule variant), papG allele I (P adhesin variant), pic (protein associated with intestinal colonization), sfa/foc (S and F1C fimbriae), and sfaS (S fimbria adhesin).

^bExPEC, extraintestinal pathogenic E. coli; PFGE, pulsed-field gel electrophoresis; Phy., phylogenetic. ExPEC was defined as the presence of ≥2 of papA and/or papC and of sfa/foc, afa/dra, kpsMII, and iutA.

^cP values (by Fisher's exact test, two-tailed) are shown where P < 0.05.

^dn.a., not applicable.

^eThe data correspond to isolates belonging to pulsotype groups consisting of ≥2 isolates or ≥5 isolates each.

^fThe denominator was 48 (rather than 50), since 2 isolates were refractory to XbaI PFGE analysis.

Several general trends were apparent from these differences (Table 3). First, statistically significant prevalence differences were more than twice as frequent in relation to ST131 status as they were in relation to CTX-M-15 status. Second, whereas most of the ST131 versus non-ST131 prevalence differences favored the ST131 isolates (e.g., for the F10 papA allele, afa/dra, iha, fimH, sat, fyuA, kpsMII, K2, K5, usp, ompT, traT, and malX), several favored the non-ST131 isolates (e.g., for hra, astA, and kpsMT III). Third, whereas quite similar sets of genes exhibited ST131-associated prevalence differences among CTX-M-15-positive compared with CTX-M-15-negative isolates, distinct sets of genes exhibited CTX-M-15-associated prevalence differences among ST131 isolates compared with non-ST131 isolates.

Consistent with the greater prevalence of many ExPEC-associated virulence genes among the ST131 isolates, a significantly greater proportion of ST131 isolates than non-ST131 isolates fulfilled molecular criteria for ExPEC, regardless of CTX-M-15 status (Table 3). Furthermore, among ST131 (but not non-ST131) isolates, CTX-M-15 was positively associated with ExPEC status. Thus, of the 4 subgroups, the CTX-M-15-positive ST131 isolates were the most likely to qualify as ExPEC (94%). Likewise, aggregate virulence scores were significantly greater among ST131 isolates compared with non-ST131 isolates and were greatest of all within the CTX-M-15-positive ST131 subgroup (see Table 5).

Table 5.

Aggregate virulence and antimicrobial resistance scores of 200 selected extended-spectrum-β-lactamase-producing Escherichia coli isolates (collected in 2000 to 2009) in relation to ST131 and bla_CTX-M-15 status

Type of score	Score median (range)				P valuea
	ST131− M15− (group 1; n = 50)	ST131− M15+ (group 2; n = 50)	ST131+ M15− (group 3; n = 50)	ST131+ M15+ (group 4; n = 50)	Group 1 vs 2	Group 3 vs 4	Group 1 vs 3	Group 2 vs 4
Virulence	4 (0-18)	4 (1-10)	10 (2-13)	10 (6-14)		0.01	< 0.001	< 0.001
Resistance	13 (4-19)	15 (7-19)	12 (4-19)	13 (9-17)	0.04			0.003

^aP values (Mann-Whitney U test) are shown where P < 0.05.

According to PCoA, which was used to summarize the total virulence gene data set for a simplified comparison among subgroups, the ST131 isolates and non-ST131 isolates were clearly separated on the coordinate 1-coordinate 2 plane (Fig. 1). In contrast, among ST131 and non-ST131 isolates alike, CTX-M-15-positive and CTX-M-negative isolates were largely intermingled, without obvious differences. AMOVA indicated that 43% of the total variance was among populations, whereas 57% was within populations.

Fig 1.

Principal coordinate analysis of virulence genotype data (upper panel) and antimicrobial resistance data (lower panel) among 200 Escherichia coli isolates (collected in 2000 to 2009) in relation to ST131 and bla_CTX-M-15 status. Populations 1 to 4 correspond to the groups shown in the tables (i.e., Table 1, non-ST131 bla_CTX-M-15 negative; Table 2, non-ST131 bla_CTX-M-15 positive; Table 3, ST131 bla_CTX-M-15 negative; Table 4, ST131 bla_CTX-M-15 positive). Upper panel (virulence genotypes): coordinates 1 and 2 capture 58% and 12% of total variation, respectively. Note the marked separation of (overlapping) populations 1 and 2 from (overlapping) populations 3 and 4. Lower panel (antimicrobial resistance): coordinates 1 and 2 capture 32% and 26% of total variation, respectively. Note the marked overlap of all four populations.

Antimicrobial resistance profiles.

Within the 200-isolate subset, resistance to each tested antimicrobial agent except imipenem was detected in ≥1 isolate (Table 4). Significant resistance prevalence differences among the 4 isolate subgroups, as defined by combined ST131 and CTX-M-15 status, were noted for 13 agents. Compared with CTX-M-15-negative isolates, CTX-M-15-positive isolates had a significantly higher prevalence of resistance to multiple β-lactam agents, regardless of ST131 status, but a significantly lower prevalence of resistance to streptomycin (if non-ST131) or sulfonamides (if ST131). In contrast, ST131 and non-ST131 isolates exhibited comparatively few significant resistance prevalence differences for β-lactam agents, but non-ST131 isolates (especially if CTX-M-15 positive) exhibited a significantly greater prevalence of resistance to numerous non-β-lactam agents (Table 4). Accordingly, aggregate resistance scores, although differing only slightly among the 4 subgroups, were highest among the CTX-M-15-positive non-ST131 isolates (median, 15) and lowest among the CTX-M-15-negative ST131 isolates (median, 12) (Table 5).

Table 4.

Antimicrobial resistance phenotypes of 200 selected extended-spectrum-β-lactamase-producing Escherichia coli isolates (collected in 2000 to 2009) in relation to ST131 and bla_CTX-M-15 status

Antimicrobial class	Specific agenta	No. (%) of isolates showing resistance to the indicated agent				P valueb
		ST131− M15− (group 1; n = 50)	ST131− M15+ (group 2; n = 50)	ST131+ M15− (group 3; n = 50)	ST131+ M15+ (group 4; n = 50)	Group 1 vs 2	Group 3 vs 4	Group 1 vs 3	Group 2 vs 4
β-Lactams	ATM	33 (66)	47 (94)	29 (58)	41 (82)	0.001	0.002
	CAZ	35 (70)	47 (94)	34 (68)	39 (78)	0.003			0.04
	CRO	44 (88)	50 (100)	39 (78)	48 (96)	0.03	0.02
	FEP	11 (22)	27 (54)	11 (22)	30 (60)	0.002	<0.001
Quinolones	NA	36 (72)	49 (98)	43 (86)	50 (100)	<0.001		0.02
	CIP	30 (60)	49 (98)	43 (86)	50 (100)	<0.001	0.01	0.006
Aminoglycosides	CN	23 (46)	31 (62)	25 (50)	17 (34)				0.009
	SF	38 (76)	20 (40)	36 (72)	26 (52)	0.001
Phenicols	C	23 (46)	14 (28)	8 (16)	2 (4)			0.002	0.002
Tetracyclines	TE	45 (90)	44 (88)	30 (60)	38 (76)			0.001
Folate antagonists	SF	44 (88)	39 (78)	39 (78)	28 (56)		0.03		0.03
	W	36 (72)	39 (78)	32 (64)	23 (46)				0.002
	SXT	37 (74)	37 (74)	31 (62)	22 (44)				0.004

^aAgents shown are those that yielded P < 0.05 in an initial four-way comparison (not shown), plus at least one pairwise comparison (as shown). These included ATM (aztreonam), C (chloramphenicol), CAZ (ceftazidime), CIP (ciprofloxacin), CN (gentamicin), CRO (ceftriaxone), FEP (cefepime), NA (nalidixic acid), S (streptomycin), SF (sulfonamide), SXT (trimethoprim-sulfamethoxazole), TE (tetracycline), and W (trimethoprim). Agents to which resistance was detected in ≥1 isolate but without significant by-group prevalence differences (definition: overall prevalence) included AMK (amikacin: 10%), AML (amoxicillin-clavulanate: 69%), AMP (ampicillin: 100%), FOX (cefoxitin: 19%), F (nitrofurantoin: 8%), KF (cefazolin: 97%), PIP (piperacillin: 96%), and TZP (piperacillin-tazobactam: 5%). No resistance was detected to IMP (imipenem).

^bP values (by Fisher's exact test, two-tailed) are shown where P < 0.05.

In a PCoA based on the 21 studied antimicrobial agents, considerable diversity of aggregate resistances profiles was evident from the overall spread of points on the coordinate 1-coordinate 2 plane (Fig. 1). However, the 4 subgroups as defined by combined CTX-M-15 status and ST131 status were considerably intermingled, without obvious among-subgroup differences. According to AMOVA, only 9% of total variance was among populations; fully 91% was within populations.

PFGE.

All but two of the 200 selected isolates yielded interpretable XbaI PFGE profiles. The 94% similarity criterion resolved 122 pulsotypes, each containing from 1 isolate (99 pulsotypes) to 27 isolates (1 pulsotype [type 968]). Twenty-three pulsotypes contained multiple isolates (99 isolates total); of these, 5 contained ≥5 isolates each (51 isolates total). Membership in a multiple-isolate pulsotype was significantly associated with both bla_CTX-M-15 (regardless of ST131 status) and ST131 (regardless of bla_CTX-M-15 status) (Table 3). Similarly, membership in a high-prevalence (≥5 isolates) pulsotype was confined to ST131 isolates, and among ST131 isolates it was significantly associated with bla_CTX-M-15 status (Table 3) and ExPEC status (92% ExPEC for high-frequency pulsotypes versus 67%; P = 0.002).

DISCUSSION

In this analysis of ESBL-positive E. coli isolates from across the United States (collected in 2000 to 2009), we defined the overall, by-center, and temporal prevalences of bla_CTX-M-15 and ST131, identified associations of these two traits with one another and with virulence genotype and resistance profiles, and assessed the clonal structures of the populations. Our findings extend the results of a previous national survey (8) and offer novel insights into the basis for the striking epidemiological success of both bla_CTX-M-15 and E. coli ST131 strains.

We found a marked predominance of both bla_CTX-M-15 and ST131 among ESBL-producing E. coli isolates collected across the United States and a very strong (albeit incomplete) association of these two traits with one another. This confirms, in a very different study population, the findings of a previous, smaller, single-year national survey (8). Notably, both bla_CTX-M-15 and ST131 occurred at each of the 15 participating centers, representing evidence of widespread distribution, but with by-center prevalence differences suggesting possible geographical or host-group specificity.

A novel finding in the present study was that both bla_CTX-M-15 and ST131 appeared to have emerged and expanded in distribution during the past decade, since they were both significantly more prevalent from 2004 onward. Likewise, both occurred among isolates from children and adults alike. The somewhat lower overall prevalences of bla_CTX-M-15 and ST131 in the present study (57% and 47%, respectively), compared with the previous national survey (78% and 67%, respectively) (8), may reflect in part the present study's inclusion of “all-comer” and pediatric isolates from 2000 through 2009, compared with the previous survey's focus on blood isolates from adults from 2007.

Our findings provide insights into possible reasons for the striking epidemiological success of bla_CTX-M-15 and ST131 and their association with one another. First, despite the obvious horizontal mobility of bla_CTX-M-15 (as demonstrated by its occurrence in diverse phylogenetic backgrounds), bla_CTX-M-15 showed evidence of clonal spread and expansion. That is, among the non-ST131 isolates, bla_CTX-M-15 was concentrated within major phylogenetic groups A and D and within high-prevalence pulsotypes within those groups. Similarly, among ST131 isolates, bla_CTX-M-15 was significantly associated with the highest-prevalence pulsotypes. To what degree bla_CTX-M-15 has contributed directly to the expansion of these lineages, or is an opportunistic hitchhiker within intrinsically successful clones, warrants further study.

Second, bla_CTX-M-15 was significantly associated with resistance to key first-line antimicrobials, notably β-lactams and fluoroquinolones, and (among non-ST131 isolates) with higher aggregate resistance scores. This predictably would favor expansion of bla_CTX-M-15-containing strains in preference to other ESBL-producing E. coli strains in the presence of selection pressure from the corresponding antimicrobials, which is abundant and likely increasing.

Third, the large molecularly inferred virulence advantage of ST131 over non-ST131 isolates (regardless of bla_CTX-M-15 status) and, within ST131, the similar (albeit smaller) advantage of bla_CTX-M-15-positive over bla_CTX-M-15-negative isolates would tend to favor expansion of ST131 and, specifically, its bla_CTX-M-15-positive subset. The marked inferred virulence advantage of ST131 strains may more than compensate for their modest resistance disadvantage compared with other ESBL-producing E. coli strains. In this regard, the high prevalence of certain ST131 pulsotypes also may relate in part to differential virulence, since isolates from high-frequency pulsotypes were significantly more likely to qualify as ExPEC than were other ST131 isolates (92% versus 67%; P = 0.0002). These highly successful lineages appear to represent an extreme version of the combined threats of virulence and resistance (22).

Study limitations included the convenience sample (with its attendant potential biases), the undefined clinical background of the isolates, and a lack of information regarding the identity of the non-CTX-M-15 ESBLs and non-ST131 clonal groups. Strengths included the large sample size relative to other studies of ESBL-positive E. coli strains from the United States, long study interval, broad geographic range, inclusion of pediatric and adult isolates, and extensive molecular characterization of the isolates.

In summary, in this large molecular-epidemiological analysis of ESBL-producing E. coli clinical isolates from the United States (collected in 2000 to 2009), we found a predominance of bla_CTX-M-15 and ST131 strains, which were widely dispersed geographically, involved pediatric as well as adult patients, and increased significantly in prevalence during the study period. Both entities were strongly associated with one another and, especially in combination, with enhanced molecularly inferred virulence, extensive antimicrobial resistance, and decreased clonal diversity, suggesting recent clonal expansion and dissemination. These findings confirm the importance in the United States of the ST131 clonal group and its characteristic bla_CTX-M-15 gene, suggest possible reasons for their emergence, and underscore the need for effective predictive and preventive measures.