We assessed Escherichia coli ST131 and its H30 and H30-Rx subclones for virulence genes, antimicrobial resistance, and extended-spectrum beta-lactamase (ESBL) type. Although both subclones were associated with ESBL production, H30-Rx isolates had higher resistance scores and were associated specifically with CTX-M-15. Three virulence genes (iha, sat, and iutA) were more prevalent among H30 than non-H30 ST131 isolates. Thus, the H30 and H30-Rx subclones are more antimicrobial resistant and have virulence profiles that are distinct from those of non-H30 ST131 isolates

Banerjee, Ritu

Johnson, James R.

Johnston, Brian D.

Kusikowski, Michael A.

Porter, Stephen

Price, Lance B.

Robicsek, Ari

Sokurenko, Evgeni

Tchesnokova, Veronika

English

PubMed

ABSTRACT
          
            We assessed
            Escherichia coli
            ST131 and its H30 and H30-Rx subclones for virulence genes, antimicrobial resistance, and extended-spectrum beta-lactamase (ESBL) type. Although both subclones were associated with ESBL production, H30-Rx isolates had higher resistance scores and were associated specifically with CTX-M-15. Three virulence genes (
            iha
            ,
            sat
            , and
            iutA
            ) were more prevalent among H30 than non-H30 ST131 isolates. Thus, the H30 and H30-Rx subclones are more antimicrobial resistant and have virulence profiles that are distinct from those of non-H30 ST131 isolates.
          </jats:p

Ritu Banerjee

Ari Robicsek

Michael A. Kuskowski

Stephen Porter

Brian D. Johnston

Evgeni Sokurenko

Veronika Tchesnokova

Lance B. Price

James R. Johnson

Crossref

Antimicrobial Agents and Chemotherapy

Molecular Epidemiology of Escherichia coli Sequence Type 131 and Its H30 and H30-Rx Subclones among Extended-Spectrum-β-Lactamase-Positive and -Negative E. coli Clinical Isolates from the Chicago Region, 2007 to 2010

George Washington University: Health Sciences Research Commons (HSRC)

Himmelfarb Health Sciences Library, The George Washington UniversityHealth Sciences Research CommonsEnvironmental and Occupational Health FacultyPublications Environmental and Occupational Health12-2013Molecular epidemiology of Escherichia colisequence type 131 and its H30 and H30-Rxsubclones among extended-spectrum-β-lactamase-positive and -negative E. coli clinical isolates fromthe Chicago region, 2007 to 2010Ritu BanerjeeMayo Clinic, Rochester, MNAri RobicsekNorthShore University Health System, Evanston, ILMichael A. KusikowskiVA Medical Center, Minneapolis, MNStephen PorterVA Medical Center, Minneapolis, MNBrian D. JohnstonVA Medical Center, Minneapolis, MNThis Journal Article is brought to you for free and open access by the Environmental and Occupational Health at Health Sciences Research Commons.It has been accepted for inclusion in Environmental and Occupational Health Faculty Publications by an authorized administrator of Health SciencesResearch Commons. For more information, please contact hsrc@gwu.edu.Recommended CitationBanerjee, R., Robicsek, A., Kuskowski, M. A., Porter, S., Johnston, B. D., Sokurenko, E., Tschesnokova, V., Price, L.B., Johnson, J. R.(2013). Molecular epidemiology of escherichia coli sequence type 131 and its H30 and H30-rx subclones among extended-spectrum-β-lactamase-positive and -negative E. coli clinical isolates from the chicago region, 2007 to 2010. Antimicrobial Agents andChemotherapy, 57(12), 6385-6388.See next page for additional authorsFollow this and additional works at: https://hsrc.himmelfarb.gwu.edu/sphhs_enviro_facpubsPart of the Environmental Public Health Commons, and the Occupational Health and IndustrialHygiene CommonsAuthorsRitu Banerjee, Ari Robicsek, Michael A. Kusikowski, Stephen Porter, Brian D. Johnston, Evgeni Sokurenko,Veronika Tchesnokova, Lance B. Price, and James R. JohnsonThis journal article is available at Health Sciences Research Commons: https://hsrc.himmelfarb.gwu.edu/sphhs_enviro_facpubs/90Molecular Epidemiology of Escherichia coli Sequence Type 131 and ItsH30 and H30-Rx Subclones among Extended-Spectrum--Lactamase-Positive and -Negative E. coli Clinical Isolates from the ChicagoRegion, 2007 to 2010Ritu Banerjee,a Ari Robicsek,b Michael A. Kuskowski,c Stephen Porter,c Brian D. Johnston,c Evgeni Sokurenko,dVeronika Tchesnokova,d Lance B. Price,e,f James R. JohnsoncMayo Clinic, Rochester, Minnesota, USAa; NorthShore University HealthSystem, Evanston, Illinois, USAb; Veterans Affairs Medical Center and University of Minnesota,Minneapolis, Minnesota, USAc; University of Washington, Seattle, Washington, USAd; Translational Genomics Research Institute, Flagstaff, Arizona, USAe; GeorgeWashington University, Washington, DC, USAfWe assessed Escherichia coli ST131 and its H30 and H30-Rx subclones for virulence genes, antimicrobial resistance, andextended-spectrum beta-lactamase (ESBL) type. Although both subclones were associated with ESBL production, H30-Rx iso-lates had higher resistance scores and were associated specifically with CTX-M-15. Three virulence genes (iha, sat, and iutA) weremore prevalent among H30 than non-H30 ST131 isolates. Thus, the H30 and H30-Rx subclones are more antimicrobial resistantand have virulence profiles that are distinct from those of non-H30 ST131 isolates.The H30 and H30-Rx subclones of Escherichia coli sequencetype 131 (ST131) have expanded more extensively than otherST131 variants, for as-yet-unexplained reasons. The H30 sub-clone, so named because it contains allele 30 of fimH (type 1 fim-brial adhesin gene), comprises almost all current fluoroquin-olone-resistant ST131 isolates (1). Within the H30 subclone, theH30-Rx subset often carries blaCTX-M-15 and may constitute itsmain repository within ST131 (2). Here we determined the prev-alence of ST131 and its H30 and H30-Rx subclones among isolatesfrom a case-control study of infections caused by extended-spec-trum--lactamase (ESBL)-producing E. coli strains (3) and com-pared these groups for virulence genotypes, antimicrobial resis-tance, and ESBL type.A total of 267 (100 ESBL-positive and 167 ESBL-negative) E.coli isolates were collected prospectively between 2007 and 2010for a case-control study, as described in detail elsewhere (3), withapproval by the NorthShore and VA Medical Center institutionalreview boards. Fifteen health care-associated isolates, collectedduring the prospective study 2 days after hospital admission,were excluded from the study reported in reference 3 but wereevaluated here. Established PCR-based methods were used to de-fine E. coli phylogenetic group (A, B1, B2, and D) (4), ST131 andits H30 subclone (1, 2, 5), major CTX-M groups (6), the presenceof blaCTX-M-15 (7), and extended virulence genotypes (8–10). TheH30-Rx subclone was identified by PCR detection of a specificsingle-nucleotide polymorphism (SNP) (G723A) within the al-lantoin-encoding gene, ybbW (2). Primers APfor63 (5=-GGTTGCGGTCTGGGCA-3=) and APrev66 (5=-CAATATCCAGCACGTTCCAGGTG-3=), with a cycling routine of 95°C for 8 min, 31 cyclesof 94°C for 20 s and 72°C for 40 s, and a final extension at 72°C (for5=), yielded a 194-bp amplicon. The resistance score was the totalReceived 24 July 2013 Returned for modification 1 September 2013Accepted 22 September 2013Published ahead of print 30 September 2013Address correspondence to Ritu Banerjee, Banerjee.ritu@mayo.edu.Copyright © 2013, American Society for Microbiology. All Rights Reserved.doi:10.1128/AAC.01604-13FIG 1 Resistance phenotypes and ESBL types among 71 ST131 isolates. (A) All71 ST131 isolates, stratified by H30 subclone status. (B) The 62 H30 subcloneisolates, stratified by H30-Rx status. ESBL, extended-spectrum -lactamase;FQ-R, fluoroquinolone resistant. Prevalence values reflect percentages of iso-lates within each indicated subgroup.December 2013 Volume 57 Number 12 Antimicrobial Agents and Chemotherapy p. 6385– 6388 aac.asm.org 6385number of agents (among 7) to which an isolate was resistant. Thevirulence score was as described previously (11). Fisher’s exact testand the Mann-Whitney U test were used for comparisons involv-ing dichotomous and continuous variables, respectively; a P valueof 0.05 was considered statistically significant. Principal coordi-nate analysis (PCoA) was used to reduce the dimensionality of themolecular data set for simplified comparisons (12).The 100 ESBL-positive (49% ST131) and 167 ESBL-negative(13% ST131; P  0.001) study isolates were predominantly fromurine (92%) and community-associated infections (95%). Themost prevalent ESBL type overall was CTX-M (84%), predomi-nantly CTX-M-15 (73%), followed by group 9 CTX-M variants(11%). CTX-M-15 was not significantly associated with ST131(data not shown).Among the 49 ESBL-positive ST131 isolates, 48 (98%) repre-sented the H30 subclone, and 44 (92%) of these represented theH30-Rx subclone. In contrast, among the 22 ESBL-negativeST131 isolates, only 14 (64%) represented the H30 subclone (P 0.001), and only 3 (14%) of these represented the H30-Rx sub-clone (P  0.001).H30 ST131 subclone isolates were overwhelmingly fluoro-quinolone resistant (98%) and ESBL positive (77%), whereas fewnon-H30 ST131 isolates were (Fig. 1A). Among H30 isolates, 94%of H30-Rx isolates, but only 27% of other H30 isolates, were ESBLpositive (Fig. 1B). Within ST131, ESBL subtype varied signifi-cantly by subclone, with CTX-M-15 being associated with H30-Rxand CTX-M-9 being associated with other H30 isolates (Fig. 1A).Antimicrobial resistance also varied by subclone, with H30subclone isolates having a higher prevalence of beta-lactam andciprofloxacin resistance (Table 1) and higher resistance scores(median, 5 versus 3; P  0.001). Similarly, among H30 isolates,H30-Rx isolates had a higher prevalence of resistance to cephalo-sporins (Table 1) and higher resistance scores (median, 6 versus 3;P  0.01).Virulence scores were significantly lower overall among ESBL-positive than ESBL-negative isolates (median, 9 versus 11; P 0.001) and among non-ST131 ESBL-positive than non-ST131ESBL-negative isolates (median, 6 versus 12; P  0.001). In con-trast, ST131 isolates had similar virulence scores (median, 10)regardless of ESBL status. Among ESBL-negative isolates, viru-lence scores were slightly lower among ST131 than non-ST131isolates (median, 10 versus 12; P  0.03). In contrast, amongESBL-positive isolates, virulence scores were much greater amongST131 than non-ST131 isolates (median, 10 versus 6; P  0.001).Within ST131, several virulence factor (VF) genes were sub-clone specific, with iha, sat, and iutA occurring predominantlyamong H30 isolates but iroN, K1, and ibeA occurring predomi-nantly among non-H30 isolates (Table 2). In contrast, among H30isolates, only kpsMII and K5 differed in prevalence betweenTABLE 1 Antimicrobial resistance by ST131 subclone among 71 Escherichia coli ST131 isolatesAntimicrobial agentWithin ST131 Within H30 ST131 subcloneNo. (%) of resistant strainsPaNo. (%) of resistant strainsPaH30 (n  62) Non-H30 (n  9) H30-Rx (n  47) Other H30 (n  15)Ampicillin 61 (98) 7 (78) 0.04 46 (98) 15 (100)Cefazolin 48 (77) 1 (11) 0.001 43 (91) 5 (33) 0.001Ceftriaxone 46 (74) 1 (11) 0.001 42 (89) 4 (27) 0.001Ceftazidime 44 (71) 1 (11) 0.001 40 (85) 4 (27) 0.001Gentamicin 23 (37) 5 (56) 15 (32) 8 (53)Ciprofloxacin 61 (98) 2 (22) 0.001 46 (98) 15 (100)TMP-SMZb 31 (50) 6 (67) 21 (45) 10 (67)a Determined by Fisher’s exact test. Values are shown where P is 0.05; for all other comparisons, P was 0.10.b TMP-SMZ, trimethoprim-sulfamethoxazole.TABLE 2 Virulence gene distribution by ST131 subclone among 71 ST131 Escherichia coli isolatesFunctional category GeneaWithin ST131 Within H30 ST131 subcloneNo. (%) of strains carrying genePbNo. (%) of strains carrying genePbH30 (n  62) Non-H30 (n  9) H30-Rx (n  47) Other H30 (n  15)Adhesin iha 60 (97) 5 (56) 0.002 45 (96) 15 (100)Toxin sat 61 (98) 3 (33) 0.001 46 (98) 15 (100)Siderophores iroN 1 (2) 2 (22) 0.04 1 (2) 0iutA 56 (90) 5 (56) 0.02 42 (89) 14 (93)Protectins kpsMII 53 (85) 8 (89) 44 (94) 9 (60) 0.004K1 2 (3) 3 (33) 0.01 1 (2) 1 (7)K5 17 (27) 1 (11) 9 (19) 8 (53) 0.02Other ibeA 0 6 (67) 0.001 0 0a Virulence genes listed are those that yielded P values of 0.05 in at least one comparison.b Determined by Fisher’s exact test. Values are shown where P is 0.05; for all other comparisons, the differences were not significant.Banerjee et al.6386 aac.asm.org Antimicrobial Agents and ChemotherapyH30-Rx and non-H30-Rx isolates (Table 2). Virulence scores weresimilar across the ST131 subclones (median for each, 10).According to PCoA, ST131 isolates had distinctive virulenceprofiles relative to non-ST131 isolates, and each subclone groupwithin ST131 (H30-Rx, other H30, and non-H30) had a charac-teristic profile (Fig. 2). Among the ST131 isolates, H30 (non-H30-Rx) profiles were most homogeneous, whereas non-H30 profileswere most diverse and overlapped most with those of non-ST131isolates (Fig. 2).Here we determined the prevalence of ST131 and its H30 andH30-Rx subclones and explored their associations with resistancephenotypes, ESBL types, and virulence profiles, among prospec-tively collected E. coli clinical isolates from the Chicago area (2007to 2010). We confirmed the well-established association betweenST131 and ESBL production (13–15), and found that the recentlyidentified H30 ST131 subclone (1, 2, 5, 16) has expanded in thestudy region more than non-H30 ST131 subclones. Furthermore,we found that within ST131, CTX-M-15 was confined almost ex-clusively to the H30-Rx subset.Our findings confirm, in a geographically distinct popula-tion, recent whole-genome-based evidence that within ST131,blaCTX-M-15 is transmitted mainly vertically within the H30-Rxlineage, after what was probably a single ancestral acquisitionevent (2). Our findings also uniquely document a continuum ofincreasing antimicrobial resistance within ST131, from the non-H30 (most susceptible) isolates to the H30-Rx isolates (most re-sistant).Furthermore, we document that, among ESBL-positive iso-lates, ST131 isolates—mostly representing the H30-Rx sub-clone— have higher virulence scores than non-ST131 isolates, im-plying greater virulence potential and thereby possibly explainingtheir high prevalence. We also identified three VF genes (iha, sat,and iutA) that are more prevalent among H30 than non-H30ST131 isolates. The mechanisms whereby specific accessory traitsmay facilitate the epidemiologic success of ST131 and its principalsubclones deserve further study.Study limitations include the fact that most isolates were fromurine and community onset infections and hence may not be rep-resentative of isolates causing invasive infections. We also lackedclinical data, so we could not correlate bacterial traits with infec-tion severity. Study strengths include the fact that that we evalu-ated a large number of ESBL-positive and ESBL-negative clinicalisolates and used novel SNP-based PCR assays to identify the re-cently recognized H30 and H30-Rx subclones within ST131.In conclusion, among ESBL-positive E. coli strains in the studyregion, the H30 ST131 subclone now accounts for almost half ofESBL-positive E. coli strains causing infections, with CTX-M-15,the most common ESBL type, being carried almost exclusively bythe H30-Rx subset within H30. Elucidation of the molecular andecologic basis for the epidemiologic success of ST131, especially itsH30 and H30-Rx components, could inform the development ofinterventions against further spread of these highly antimicrobial-resistant lineages.ACKNOWLEDGMENTSThis material is based upon work supported by Office of Research andDevelopment, Medical Research Service, Department of Veterans Affairs,grant 1 I01 CX000192 01 (J.R.J.); NIH grants RC4-AI092828 (E.V.S. andJ.R.J.) and 2KL2RR024151-07 (R.B.); and a research grant fromMerck, Inc.J.R.J. has received grants, contracts, or consultancies from ICET,Rochester Medical, and Syntiron. J.R.J., L.B.P., and E.V.S. have patentapplications related to diagnostic tests for E. coli clonal groups.REFERENCES1. Johnson J, Tchesnokova V, Johnston B, Clabots C, Roberts P, Billig M,Riddell K, Rogers P, Qin X, Butler-Wu S, Price L, Aziz M, Nicolas-Chanoine M, Debroy C, Robicsek A, Hansen G, Urban C, Platell J,Trott D, Zhanel G, Weissman S, Cookson B, Fang F, Limaye A, ScholesD, Chattopadhyay S, Hooper D, Sokurenko E. 2013. Abrupt emergenceof a single dominant multidrug-resistant strain of Escherichia coli. J. Infect.Dis. 207:919 –928.2. Price L, Johnson J, Aziz M, Clabots C, Johnston B, Tchesnokova V,Nordstrom L, Pearson T, Keim P, Sokurenko E. Epidemic clonal ex-pansion of CTX-M-15-producing Escherichia coli ST131. mBio, in press.3. Banerjee R, Strahilevitz J, Johnson J, Nagwekar P, Schora D, Shevrin I,Du H, Peterson L, Robicsek A. 2013. Predictors and molecular epidemi-ology of community-onset extended-spectrum beta-lactamase (ESBL)FIG 2 Principal coordinate analysis (PCoA) of virulence gene profiles among 267 Escherichia coli isolates. The PCoA was based on results for all 50 virulencegenes studied. Each isolate is plotted based on its values for PCoA coordinates 1 (x axis) and 2 (y axis), which collectively capture 64.5% of total variance in thedata set. For ST131, 47 H30-Rx, 15 other H30, and 9 non-H30 isolates were evaluated; for non-ST131, 51 ESBL-producing and 145 non-ESBL-producing isolateswere evaluated.E. coli ST131 and Its H30 and H30-Rx SubclonesDecember 2013 Volume 57 Number 12 aac.asm.org 6387Escherichia coli infection in a Midwestern community Infect. ControlHosp. Epidemiol. 34:947–953.4. Clermont O, Bonacorsi S, Bingen E. 2000. Rapid and simple determina-tion of Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66:4555– 4558.5. Colpan A, Porter S, Johnston B, Clabots C, Anway R, Thao L, Kus-kowski MA, Tchesnokova V, Sokurenko EV, Johnson J. Escherichia colisequence type 131 (ST131) subclone H30 as an emergent multidrug-resistant pathogen among U.S. veterans. Clin. Infect. 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Epidemic clonal groups of Escherichia coli as a cause of antimicro-bial-resistant urinary tract infections in Canada, 2002–2004. Antimicrob.Agents Chemother. 53:2733–2739.10. Johnson J, Stell A. 2000. Extended virulence genotypes of Escherichia colistrains from patients with urosepsis in relation to phylogeny and hostcompromise. J. Infect. Dis. 181:261–272.11. Johnson J, Murray A, Gajewski A, Sullivan M, Snippes P, Kuskowski M,Smith K. 2003. Isolation and molecular characterization of nalidixic acid-resistant extraintestinal pathogenic Escherichia coli from retail chickenproducts. Antimicrob. Agents Chemother. 47:2161–2168.12. Peakall R, Smouse P. 2006. GENALEX 6: genetic analysis in Excel: pop-ulation genetic software for teaching and research. Mol. Ecol. Notes6:288 –295.13. Olesen B, Hansen D, Nilsson F, Frimodt-Møller J, Leihof R, Struve C,Scheutz F, Johnston B, Krogfelt K, Johnson J. 2013. Prevalence andcharacteristics of the epidemic multiresistant Escherichia coli ST131 clonalgroup among extended-spectrum beta-lactamase-producing E. coli iso-lates in Copenhagen, Denmark. J. Clin. Microbiol. 51:1779 –1785.14. Pitout JDD, Campbell L, Church DL, Gregson DB, Laupland KB. 2009.Molecular characteristics of travel-related extended-spectrum beta-lactamase-producing Escherichia coli isolates from the Calgary health re-gion. Antimicrob. Agents Chemother. 53:2539 –2543.15. Blanco J, Mora A, Mamani R, López C, Blanco M, Dahbi G, Herrera A,Marzoa J, Fernández V, de la Cruz F, Martínez-Martínez L, Alonso MP,Nicolas-Chanoine M-H, Johnson JR, Johnston B, López-Cerero L,Pascual A, Rodríguez-Baño J, the Spanish Group for Nosocomial In-fections (GEIH). 2013. Four main virotypes among extended-spectrum-lactamase-producing isolates of Escherichia coli O25b:H4-B2-ST131:bacterial, epidemiological, and clinical characteristics. J. Clin. Microbiol.51:3358 –3367.16. Tchesnokova V, Billig M, Chattapadhyay S, Linardopoulou E, AprikianP, Roberts PL, Skrivankova PV, Johnston B, Gileva A, Igusheva I,Toland A, Riddell K, Rogers P, Qin X, Bulter-Wu S, Cookson BT, FangFC, Kahl B, Price LB, Weissman SJ, Limaye A, Scholes D, Johnson JR,Sokurenko EV. 2013. Predictive diagnostics for Escherichia coli infectionsbased on the clonal association of antimicrobial resistance and clinicaloutcome. J. Clin. Microbiol. 51:2991–2999.Banerjee et al.6388 aac.asm.org Antimicrobial Agents and Chemotherapy

Molecular epidemiology of Escherichia coli sequence type 131 and its H30 and H30-Rx subclones among extended-spectrum-β-lactamase-positive and -negative E. coli clinical isolates from the Chicago region, 2007 to 2010

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Molecular epidemiology of Escherichia coli sequence type 131 and its H30 and H30-Rx subclones among extended-spectrum-β-lactamase-positive and -negative E. coli clinical isolates from the Chicago region, 2007 to 2010

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