CTX-M-123, a Novel Hybrid of the CTX-M-1 and CTX-M-9 Group β-Lactamases Recovered from Escherichia coli Isolates in China

The chimeric bla_CTX-M-123 gene was identified in two ceftazidime-resistant Escherichia coli isolates from animals in different Chinese provinces. Like other CTX-M-1/9 group hybrids (CTX-M-64 and CTX-M-132), the ends (amino acids 1 to 135 and 234 to 291) of CTX-M-123 match CTX-M-15 while the central part (122 to 241) matches CTX-M-14. bla_CTX-M-123 is carried on related, but not identical, ∼90-kb IncI1 plasmids in the two isolates, and one isolate simultaneously carries the group 1 bla_CTX-M-55 gene on an additional IncI2 plasmid.     

Detection and Molecular Characterization of Escherichia coli CTX-M-15 and Klebsiella pneumoniae SHV-12 β-Lactamases from Bovine Mastitis Isolates in the United Kingdom

Recent reports raised concerns about the role that farm stock may play in the dissemination of extended-spectrum β-lactamase (ESBL)-producing bacteria. This study characterized the ESBLs in two Escherichia coli and three Klebsiella pneumoniae subsp. pneumoniae isolates from cases of clinical bovine mastitis in the United Kingdom. Bacterial culture and sensitivity testing of bovine mastitic milk samples identified Gram-negative cefpodoxime-resistant isolates, which were assessed for their ESBL phenotypes. Conjugation experiments and PCR-based replicon typing (PBRT) were used for characterization of transferable plasmids. E. coli isolates belonged to sequence type 88 (ST88; determined by multilocus sequence typing) and carried bla_CTX-M-15 and bla_TEM-1, while K. pneumoniae subsp. pneumoniae isolates carried bla_SHV-12 and bla_TEM-1. Conjugation experiments demonstrated that bla_CTX-M-15 and bla_TEM-1 were carried on a conjugative plasmid in E. coli, and PBRT identified this to be an IncI1 plasmid. The resistance genes were nontransferable in K. pneumoniae subsp. pneumoniae isolates. Moreover, in the E. coli isolates, an association of ISEcp1 and IS26 with bla_CTX-M-15 was found where the IS26 element was inserted upstream of both ISEcp1 and the bla_CTX-M promoter, a genetic arrangement highly similar to that described in some United Kingdom human isolates. We report the first cases in Europe of bovine mastitis due to E. coli CTX-M-15 and also of bovine mastitis due to K. pneumoniae subsp. pneumoniae SHV-12 β-lactamases in the United Kingdom. We also describe the genetic environment of bla_CTX-M-15 and highlight the role that IncI1 plasmids may play in the spread and dissemination of ESBL genes, which have been described in both human and cattle isolates.     

New Variant of CTX-M-Type Extended-Spectrum β-Lactamases,CTX-M-71, with a Gly238Cys Substitution in a Klebsiella pneumoniae Isolate from Bulgaria

A single Klebsiella pneumoniae strain isolated in a Bulgarian hospital was found to produce CTX-M-71, a new CTX-M variant characterized by one amino acid substitution from glycine to cysteine at position 238 in comparison to CTX-M-15. This exchange decreased the hydrolytic activity of the β-lactamase for cefotaxime, ceftazidime, and cefepime.Since the first reports on CTX-M-type extended-spectrum β-lactamases (ESBLs) in the late 1980s and early 1990s (2, 4, 13), their number has increased to 89 (http://www.lahey.org/studies [last accessed in June 2009]) and they have become the most prevalent ESBL-type worldwide (6).Several amino acid residues of CTX-M β-lactamases have been investigated with respect to their influence on hydrolytic activity. Asn104, Asn132, and Ser237 are thought to fix the cefotaxime substrate within the binding site (5). Arg276 appears to be important for the hydrolysis of cefotaxime, and the substitutions Asp240Gly and Pro167Ser improve the hydrolytic activity of CTX-M enzymes for ceftazidime (5, 17).We analyzed a Klebsiella pneumoniae isolate producing a new CTX-M variant, CTX-M-71, with one amino acid substitution (Gly238Cys) in comparison to CTX-M-15.K. pneumoniae AH24-270 was isolated in September 2003 from the urine of a 29-year-old male patient at the Alexandrovska University Hospital in Sofia, Bulgaria. The patient had been hospitalized since June 2003 due to cranial brain trauma, aspiration pneumonia, and respiratory dysfunction. Antibiotic treatment prior to isolation of the strain included penicillin, metronidazole, amikacin, vancomycin, meropenem, ceftazidime, and piperacillin-tazobactam.The sequencing of the bla_CTX-M gene of K. pneumoniae AH24-270, by using the oligonucleotides CTX-M-1/P1c (5′-TCGTCTCTTCCAGAATAAGG-3′) and CTX-M-1/P2c (5′-AAGGAGAACCAGGAACCACG-3′), revealed one nucleotide exchange from G to T at open reading frame position 721, causing an amino acid substitution from glycine to cysteine at position 238 in comparison to CTX-M-15 (Ambler numbering [1]). This new CTX-M variant was named CTX-M-71. So far, only one other natural CTX-M β-lactamase, namely, CTX-M-34 (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY515297","term_id":"41079306","term_text":"AY515297"}}AY515297), which also harbored a cysteine at position 238 was described (15). All other CTX-M variants carry a glycine at that position. However, Shimizu-Ibuka et al. introduced in vitro the Gly238Cys exchange in Toho-1 (CTX-M-44) (19).Conjugative plasmid transfer, performed as described previously (11), located bla_CTX-M-71 on a transferable plasmid together with determinants for resistance to tobramycin, gentamicin, and tetracycline (data not shown).The MICs, determined by an agar dilution procedure following Clinical and Laboratory Standards Institute (formerly NCCLS) guidelines (16), are shown in Table ​Table1.1. Interestingly, K. pneumoniae AH24-270 was resistant to ertapenem and had an elevated MIC for meropenem but was susceptible to imipenem. The MICs for meropenem and ertapenem, but not for imipenem and faropenem, were lowered in combination with clavulanic acid. The transconjugant showed no elevated MICs for carbapenems.

TABLE 1.

Antimicrobial susceptibilities of wild-type, transconjugant, and transformant strains

High Prevalence of ST131 Isolates Producing CTX-M-15 and CTX-M-14 among Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolates from Canada

Phenotypic and genotypic methods were used to characterize extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli isolated in 2007 from 11 different Canadian medical centers. Of the 209 ESBL-producing E. coli isolates tested, 148 (71%) produced CTX-M-15, 17 (8%) produced CTX-M-14, 5 (2%) produced CTX-M-3, and 1 produced CTX-M-27. Overall, 96 (46%) of the ESBL producers belonged to clonal complex ST131, with the highest prevalence in Brampton, Calgary, and Winnipeg. ST131 is an important cause of community onset urinary tract infections due to ESBL-producing E. coli across Canada.Since 2000, Escherichia coli producing CTX-M enzymes has emerged worldwide as an important cause of community onset urinary tract infections (UTIs), and this has been called “the CTX-M pandemic” (3). This phenomenon accelerated rapidly, especially during the past 5 years, and today organisms producing these enzymes are the most common type of extended-spectrum β-lactamase (ESBL) producers found in most areas of the world (24). Although several members of the family Enterobacteriaceae that produce CTX-M β-lactamases have been involved in hospital-acquired infections, E. coli producing these enzymes is more likely to be responsible for community onset infections (21).Currently, the most widely distributed CTX-M enzyme is CTX-M-15, which was first detected in E. coli from India in 2001 (10). Multidrug-resistant, CTX-M-15-producing E. coli is emerging worldwide, especially since 2003, as an important pathogen causing both community onset and hospital-acquired infections (6, 14, 20).Two recent studies using multilocus sequencing typing (MLST) identified a single clone of CTX-M-15-producing E. coli, named ST131, in isolates from several countries, including Spain, France, Canada, Portugal, Switzerland, Lebanon, India, Kuwait, and Korea (6, 14). This clone is associated with serogroup O25, belongs to highly virulent phylogenetic group B2, and harbors multidrug-resistant IncFII plasmids. Since those initial studies, isolates of clonal complex ST131 that produce CTX-M-15 have also been reported in several countries, including the United Kingdom (11), Italy (2), Turkey (27), Croatia (12), Japan (25), the United States (8), and Norway (13). Isolates of clonal complex ST131 have also been associated with other types of β-lactamases, as well as ciprofloxacin-resistant E. coli isolates that do not have ESBLs (4, 9, 12, 15).Due to the worldwide emergence of clone ST131 isolates that produce CTX-M β-lactamases, we designed a study to investigate the prevalence and characteristics of this clone in ESBL-producing E. coli isolated from community and hospital settings during 2007 from 11 different Canadian medical centers.(This study was presented at the 26th International Congress of Chemotherapy and Infection in Toronto, Ontario, Canada, 2009 [abstract P179].)Nonrepeat ESBL-producing E. coli was collected over a 1-month period in 2007 from different Canadian medical centers representing 11 cities in six provinces (Table ​(Table1).1). ESBL production was confirmed phenotypically by using the Clinical and Laboratory Standards Institute [CLSI] criteria for ESBL screening and disk confirmation tests (5).

TABLE 1.

ESBL-producing E. coli isolated at various medical centers in Canada

Conformational Change Observed in the Active Site of Class C β-Lactamase MOX-1 upon Binding to Aztreonam

We solved the crystal structure of the class C β-lactamase MOX-1 complexed with the inhibitor aztreonam at 1.9Å resolution. The main-chain oxygen of Ser315 interacts with the amide nitrogen of aztreonam. Surprisingly, compared to that in the structure of free MOX-1, this main-chain carboxyl changes its position significantly upon binding to aztreonam. This result indicates that the interaction between MOX-1 and β-lactams can be accompanied by conformational changes in the B3 β-strand main chain.     

Hydrolysis and Inhibition Profiles of β-Lactamases from Molecular Classes A to D with Doripenem,Imipenem, and Meropenem

The stability of doripenem to hydrolysis by β-lactamases from molecular classes A to D was compared to the stability for imipenem and meropenem. Doripenem was stable to hydrolysis by extended-spectrum β-lactamases and AmpC type β-lactamases and demonstrated high affinity for the AmpC enzymes. For the serine carbapenemases SME-3 and KPC-2 and metallo-β-lactamases IMP-1 and VIM-2, doripenem hydrolysis was generally 2- to 150-fold slower than imipenem hydrolysis. SPM-1 hydrolyzed meropenem and doripenem fourfold faster than imipenem.Doripenem is a parenteral carbapenem with broad-spectrum activity against many aerobic and anaerobic pathogens. Doripenem MICs against gram-negative clinical isolates are frequently ≤0.5 μg/ml, even in Enterobacteriaceae expressing extended-spectrum β-lactamases (ESBLs) or overproduced AmpC (7, 12, 14). Resistance to doripenem and other carbapenems is observed in isolates producing metallo-β-lactamases (MBLs) or class A or class D carbapenemases (12, 13). In this study, we evaluated the hydrolysis of doripenem by a spectrum of β-lactamases from almost every functional group (3) and compared the doripenem kinetic profiles to those obtained for imipenem and meropenem.(These data were presented in part as poster P909 at the 18th European Congress of Clinical Microbiology and Infectious Diseases, Barcelona, Spain, 2008.)The broth microdilution methodology was used to determine MICs (6). Doripenem was from Shionogi & Co., Ltd. (Hyogo, Japan). Benzylpenicillin and cephaloridine were from Sigma (St. Louis, MO). Ceftazidime, imipenem, and meropenem were from U.S. Pharmacopeia (Rockville, MD).MICs are shown in Table ​Table11 for the strains used as sources of β-lactamases. The carbapenem MICs for isolates producing broad-spectrum, extended-spectrum, and AmpC β-lactamases were ≤2 μg/ml across the represented bacteria. Doripenem and meropenem MICs for the non-carbapenemase-producing isolates were generally four- to eightfold lower than those obtained for imipenem. Reduced susceptibility was apparent in isolates that expressed β-lactamases with known carbapenem hydrolysis profiles.

TABLE 1.

MICs for strains expressing characterized β-lactamases^b

Molecular and Biochemical Characterization of CTX-M-131, a Natural Asp240Gly Variant Derived from CTX-M-2, Produced by a Providencia rettgeri Clinical Strain in S?o Paulo,Brazil

CTX-M-131 is a natural Asp240Gly variant from the CTX-M-2 group detected in a Providencia rettgeri clinical strain from Brazil. Molecular analysis showed that bla_CTX-M-131 was inserted in a complex class 1 integron harbored by a 112-kb plasmid, which has not been previously described as a platform for CTX-M-encoding genes with the Asp240Gly mutation. Steady-state kinetic parameters showed that the enzyme has a typical cefotaximase catalytic profile and an enhanced activity against ceftazidime.     

Activity of Ceftazidime-Avibactam against Extended-Spectrum- and AmpC β-Lactamase-Producing Enterobacteriaceae Collected in the INFORM Global Surveillance Study from 2012 to 2014

The in vitro activity of ceftazidime-avibactam was evaluated against 34,062 isolates of Enterobacteriaceae from patients with intra-abdominal, urinary tract, skin and soft-tissue, lower respiratory tract, and blood infections collected in the INFORM (International Network For Optimal Resistance Monitoring) global surveillance study (176 medical center laboratories in 39 countries) in 2012 to 2014. Overall, 99.5% of Enterobacteriaceae isolates were susceptible to ceftazidime-avibactam using FDA approved breakpoints (susceptible MIC of ≤8 μg/ml; resistant MIC of ≥16 μg/ml). For individual species of the Enterobacteriaceae, the ceftazidime-avibactam MIC inhibiting ≥90% of isolates (MIC₉₀) ranged from 0.06 μg/ml for Proteus species to 1 μg/ml for Enterobacter spp. and Klebsiella pneumoniae. Carbapenem-susceptible isolates of Escherichia coli, K. pneumoniae, Klebsiella oxytoca, and Proteus mirabilis with a confirmed extended-spectrum β-lactamase (ESBL) phenotype, or a ceftazidime MIC of ≥16 μg/ml if the ESBL phenotype was not confirmed by clavulanic acid inhibition, were characterized further to identify the presence of specific ESBL- and plasmid-mediated AmpC β-lactamase genes using a microarray-based assay and additional PCR assays. Ceftazidime-avibactam demonstrated potent activity against molecularly confirmed ESBL-producing (n = 5,354; MIC₉₀, 0.5 μg/ml; 99.9% susceptible), plasmid-mediated AmpC-producing (n = 246; MIC₉₀, 0.5 μg/ml; 100% susceptible), and ESBL- and AmpC-producing (n = 152; MIC₉₀, 1 μg/ml; 100% susceptible) isolates of E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis. We conclude that ceftazidime-avibactam demonstrates potent in vitro activity against globally collected clinical isolates of Enterobacteriaceae, including isolates producing ESBLs and AmpC β-lactamases.     

Mobile Genetic Elements Related to the Diffusion of Plasmid-Mediated AmpC β-Lactamases or Carbapenemases from Enterobacteriaceae: Findings from a Multicenter Study in Spain

We examined the genetic context of 74 acquired ampC genes and 17 carbapenemase genes from 85 of 640 Enterobacteriaceae isolates collected in 2009. Using S1 pulsed-field gel electrophoresis and Southern hybridization, 37 of 74 bla_AmpC genes were located on large plasmids of different sizes belonging to six incompatibility groups. We used sequencing and PCR mapping to investigate the regions flanking the acquired ampC genes. The bla_CMY-2-like genes were associated with ISEcp1; the surrounding bla_DHA genes were similar to Klebsiella pneumoniae plasmid pTN60013 associated with IS26 and the psp and sap operons; and the bla_ACC-1 genes were associated with IS26 elements inserted into ISEcp1. All of the carbapenemase genes (bla_VIM-1, bla_IMP-22, and bla_IMP-28) were located in class 1 integrons. Therefore, although plasmids are the main cause of the rapid dissemination of ampC genes among Enterobacteriaceae, we need to be aware that other mobile genetic elements, such as insertion sequences, transposons, or integrons, can be involved in the mobilization of these genes of chromosomal origin. Additionally, three new integrons (In846 to In848) are described in this study.     

Contemporary Diversity of β-Lactamases among Enterobacteriaceae in the Nine U.S. Census Regions and Ceftazidime-Avibactam Activity Tested against Isolates Producing the Most Prevalent β-Lactamase Groups

Escherichia coli (328 isolates), Klebsiella pneumoniae (296), Klebsiella oxytoca (44), and Proteus mirabilis (33) isolates collected during 2012 from the nine U.S. census regions and displaying extended-spectrum-β-lactamase (ESBL) phenotypes were evaluated for the presence of β-lactamase genes, and antimicrobial susceptibility profiles were analyzed. The highest ESBL rates were noted for K. pneumoniae (16.0%, versus 4.8 to 11.9% for the other species) and in the Mid-Atlantic and West South Central census regions. CTX-M group 1 (including CTX-M-15) was detected in 303 strains and was widespread throughout the United States but was more prevalent in the West South Central, Mid-Atlantic, and East North Central regions. KPC producers (118 strains [112 K. pneumoniae strains]) were detected in all regions and were most frequent in the Mid-Atlantic region (58 strains). Thirteen KPC producers also carried bla_CTX-M. SHV genes encoding ESBL activity were detected among 176 isolates. Other β-lactamase genes observed were CTX-M group 9 (72 isolates), FOX (10), TEM ESBL (9), DHA (7), CTX-M group 2 (3), NDM-1 (2 [Colorado]), and CTX-M groups 8 and 25 (1). Additionally, 62.9% of isolates carried ≥2 β-lactamase genes. KPC producers were highly resistant to multiple agents, but ceftazidime-avibactam (MIC_50/90, 0.5/2 μg/ml) and tigecycline (MIC_50/90, 0.5/1 μg/ml) were the most active agents tested. Overall, meropenem (MIC₅₀, ≤0.06 μg/ml), ceftazidime-avibactam (MIC₅₀, 0.12 to 0.5 μg/ml), and tigecycline (MIC₅₀, 0.12 to 2 μg/ml) were the most active antimicrobials when tested against this collection. NDM-1 producers were resistant to all β-lactams tested. The diversity and increasing prevalence of β-lactamase-producing Enterobacteriaceae have been documented, and ceftazidime-avibactam was very active against the vast majority of β-lactamase-producing strains isolated from U.S. hospitals.     

First Detection of CTX-M and SHV Extended-Spectrum β-Lactamases in Escherichia coli Urinary Tract Isolates from Dogs and Cats in the United States

One hundred fifty canine and feline Escherichia coli isolates associated with urinary tract infections were screened for the presence of extended-spectrum β-lactamase (ESBL) genes. Out of 60 isolates suspected to be ESBL positive based on antimicrobial susceptibility testing, 11 ESBLs were identified, including one SHV-12 gene, one CTX-M-14 gene, and nine CTX-M-15 genes. This study provides the first report of CTX-M- and SHV-type ESBLs in dogs and cats in the United States.The first detection of an extended-spectrum β-lactamase (ESBL) in an organism from an animal was reported in Japan in 1988 in an Escherichia coli isolate from a laboratory dog (13). Since that time, numerous reports of ESBL-positive isolates from dogs and cats, as well as from other animal species (5), have been made worldwide (4, 8, 11, 17, 25, 26). Only one study has identified the presence of an ESBL in isolates from animals in the United States, i.e., in Salmonella enterica serovar Newport from horses (21). We hypothesized that ESBL genes would be present in urinary E. coli isolates from companion animals in the United States. The purpose of this study was to screen a group of 150 E. coli isolates from canine and feline patients that had been diagnosed with a urinary tract infection (UTI) for the presence of ESBL genes.A convenience sample of 150 E. coli isolates collected from canine and feline patients at the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania with clinical signs or evidence on routine urinalysis of a UTI between 1 September 2004 and 31 December 2007 was used in this study. Isolates were frozen in Microbank tubes (ProLab Diagnostics, Austin, TX) and stored at −70°C prior to use.MICs were determined using a Negative Combo 31 panel on a MicroScan Walkaway 40 (Dade Behring, Siemens Healthcare Diagnostics, Deerfield, IL). Results were interpreted using Clinical and Laboratory Standards Institute (CLSI) breakpoints (7). Isolates collected from the same individual animal within a 45-day period were considered to be the same strain, and only the first isolate collected was analyzed (subsequent isolates were considered redundant). All isolates with a cefpodoxime MIC of ≥4 μg/ml and a ceftazidime MIC of ≥1 μg/ml were identified as an “ESBL alert” on the MicroScan Walkaway. ESBL confirmatory testing was performed via the Etest method using ceftazidime-ceftazidime-clavulanic acid and cefotaxime-cefotaxime-clavulanic acid strips in accordance with CLSI guidelines (7).Due to the high prevalence of cefoxitin resistance in this population, PCR was performed to detect the presence of a bla_AmpC gene, the product of which can mask the effects of clavulanic acid on the ESBL confirmatory test (18, 27). Since the primers used in this study to identify the bla_AmpC gene (18) have since been shown to amplify the plasmid-mediated bla_CMY gene from Citrobacter freundii (19), it can be inferred that the genes detected were part of the bla_CMY lineage. Salmonella Newport strain 0007-33 was used as the positive control (21). PCR was performed for the genes bla_TEM, bla_SHV, and bla_CTX-M as published previously (9, 10, 27). Salmonella Newport strain 0007-33 was also used as the TEM and SHV positive control (21), and E. coli strain MISC 336 (CTX-M-1 positive) was used as the CTX-M positive control.The bla_TEM, bla_SHV, and bla_CTX-M PCR products were sequenced using both strands of DNA for each PCR product. Protein sequences were aligned using Lasergene software (DNASTAR, Inc., Madison, WI) and included GenBank sequences (http://www.ncbi.nlm.nih.gov/GenBank/index.html) to confirm ESBL genotype. Mutations were evaluated with reference to the Lahey Clinic website (http://www.lahey.org/studies/). GenBank accession numbers used for alignment of protein sequences were {"type":"entrez-protein","attrs":{"text":"AAR25033","term_id":"38606069","term_text":"AAR25033"}}AAR25033 for TEM-1 and {"type":"entrez-protein","attrs":{"text":"ABF29674","term_id":"94502886","term_text":"ABF29674"}}ABF29674 for SHV-2. CTX-M accession numbers were derived from a list on the Lahey Clinic website. Specific primers for the CTX-M-1 group (M13U and M13L) were used to amplify the entire coding sequences of these bla_CTX-M genes (23). Sequencing and analysis were carried out as described above to identify the specific CTX-M subtype.Seventy of the 150 E. coli isolates had an “ESBL alert” on the MicroScan Walkaway, and after removal of redundant isolates, 60 isolates were tested further. ESBL confirmatory testing was positive for six of these 60 isolates (Table ​(Table1,1, column 3).

TABLE 1.

Distribution of β-lactamase and extended-spectrum β-lactamase genes

Effects of Klebsiella pneumoniae Carbapenemase Subtypes,Extended-Spectrum β-Lactamases,and Porin Mutations on the In Vitro Activity of Ceftazidime-Avibactam against Carbapenem-Resistant K. pneumoniae

Avibactam is a novel β-lactamase inhibitor with affinity for Klebsiella pneumoniae carbapenemases (KPCs). In combination with ceftazidime, the agent demonstrates activity against KPC-producing K. pneumoniae (KPC-Kp). KPC-Kp strains are genetically diverse and harbor multiple resistance determinants, including defects in outer membrane proteins and extended-spectrum β-lactamases (ESBLs). Mutations in porin gene ompK36 confer high-level carbapenem resistance to KPC-Kp strains. Whether specific mechanisms of antimicrobial resistance also influence the activity of ceftazidime-avibactam is unknown. We defined the effects of ceftazidime-avibactam against 72 KPC-Kp strains with diverse mechanisms of resistance, including various combinations of KPC subtypes and ESBL and ompK36 mutations. Ceftazidime MICs ranged from 64 to 4,096 μg/ml and were lowered by a median of 512-fold with the addition of avibactam. All strains exhibited ceftazidime-avibactam MICs at or below the CLSI breakpoint for ceftazidime (≤4 μg/ml; range, 0.25 to 4). However, the MICs were within two 2-fold dilutions of the CLSI breakpoint against 24% of the strains, and those strains would be classified as nonsusceptible to ceftazidime by EUCAST criteria (MIC > 1 μg/ml). Median ceftazidime-avibactam MICs were higher against KPC-3 than KPC-2 variants (P = 0.02). Among KPC-2-Kp strains, the presence of both ESBL and porin mutations was associated with higher drug MICs compared to those seen with either factor alone (P = 0.003 and P = 0.02, respectively). In conclusion, ceftazidime-avibactam displays activity against genetically diverse KPC-Kp strains. Strains with higher-level drug MICs provide a reason for caution. Judicious use of ceftazidime-avibactam alone or in combination with other agents will be important to prevent the emergence of resistance.     

Complete nucleotide sequence of plasmid pTN48, encoding the CTX-M-14 extended-spectrum β-lactamase from an Escherichia coli O102-ST405 strain

The sequence of pTN48, a plasmid of the FII-FIB replicon type that encodes a CTX-M-14 enzyme in an Escherichia coli strain of the phylogenetic group D₂ O102-ST405 clone, was determined. pTN48 is, for the most part, a mosaic of virulence, antibiotic resistance, and addiction system modules found in various other plasmids. The presence of multiple addiction systems indicates that the plasmid should be stably maintained in the E. coli clone, favoring dissemination of the CTX-M-14 enzyme.The epidemiology of extended-spectrum beta-lactamases (ESBLs) has drastically changed in recent years. An explosive spread of CTX-M-type enzymes, with Escherichia coli as the main host, has occurred in both hospital and community settings worldwide (4, 23). Two phenomena may explain such an epidemic profile: the spread of plasmids bearing antibiotic resistance genes between bacterial strains and the spread of bacterial clones bearing resistance-encoding plasmids. Recently, the application of multilocus sequence typing revealed that a few E. coli clones with the ability to capture a large panel of ESBLs have disseminated internationally (5, 7, 14, 16, 18, 19). Furthermore, it has been shown that two of these clones, the O25b sequence type 131 (ST131) clone of the B2 phylogenetic group and the O102-ST405 clone of the D₂ phylogenetic group, were highly virulent in a mouse model of septicemia (6, 16). The dissemination of such resistant and virulent clones constitutes a major public health concern and prompted us to examine ESBL-encoding plasmids associated with these clones. We therefore sequenced pTN48, a nonconjugative plasmid of the FII-FIB replicon type carrying a CTX-M-14 ESBL gene and originating from a strain of the E. coli D₂ phylogenetic subgroup I O102-ST405 clone (8). Strain TN48 was isolated from an adult patient with a urinary tract infection in 2004 in Paris and was resistant to ciprofloxacin, streptomycin, kanamycin, gentamicin, tobramycin, netilmicin, tetracycline, nalidixic acid, chloramphenicol, trimethoprim, and sulfonamides; the DH10B electroporant containing pTN48 (DH10B/pTN48) displayed the same resistance phenotype, except that it remained susceptible to ciprofloxacin.The plasmid DNA was extracted from the electroporant (8) by using the Qiagen Large Construct kit (Qiagen, Courtaboeuf, France), and Solexa technology was used for sequencing. The reads generated were assembled de novo into 38 contigs with VELVET software (28). Combinatorial PCRs were used to assemble the contigs and to fill in gaps. MaGe (Magnifying Genomes) software was used for gene annotation and comparative analysis as described elsewhere (26). Manual validation of the automatic annotation was performed using the MaGe interface.Plasmid pTN48 is a circular molecule of 165,657 bp (overall G+C content of 50.21%) harboring 194 predicted open reading frames (ORFs); 134 were assigned known functions. Twelve unique ORFs corresponding to about 18,000 bp had no homolog in public databases and thus could be considered specific to this plasmid. Thirty-six coding sequences as part of 25 insertion sequences, in particular, IS1 and IS26, were found throughout the plasmid. pTN48 can be divided into three functional modules that are involved in antimicrobial resistance, virulence, and plasmid transfer and maintenance (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Circular representation of plasmid pTN48. The circles display (from the outside) (i) GC percent deviation (GC window − mean GC) in a 1,000-bp window, (ii) predicted coding sequences transcribed in a clockwise direction, (iii) predicted coding sequences transcribed in a counterclockwise direction, (iv) GC skew ([G + C]/[G − C]) in a 1,000-bp window, and (v) coordinates in kilobase pairs from the origin of replication. The color code for the various gene functions is shown below the map.Antibiotic resistance is encoded within a continuous region of 42,794 bp (Fig. ​(Fig.2)2) divided into six subregions sharing strong homology with different plasmids by five copies of IS26 (the third one being truncated). The first subregion of 1,600 bp encompassed bla_TEM-1b associated with a remnant of transposon Tn2. The second subregion, a 12,000-bp region composed of a class 1 integron (dfrA17, aadA5, qacEΔ1, sul1), chrA, padR, IS6100, mphA, mrx, and mphR showed >99% similarity to a CTX-M-15-encoding plasmid, pEK499 (27). The third subregion, a 9,800-bp sequence, showed >99% similarity to a Klebsiella pneumoniae CTX-M-19-encoding plasmid, pILT-3 (24). It included orf1 of Tn1721, into which were inserted bla_CTX-M-14 and its environment (IS903 and ISEcp1B), and a second class 1 integron, in inverted orientation with respect to the previous one, containing aacA4 and cmlA. The next subregion, a 3,200-bp sequence containing aac(3)-II, was found to be identical to a CTX-M-15-encoding plasmid, pC15-1a (2). The fifth subregion was a 5,000-bp sequence containing an ISCR14-like element associated with truncated groEL and ermBC (3, 25). Finally, the last subregion, an 11,000-bp sequence, can be divided into two parts, a first part containing repA and repC of the repABC operon, an antisense RNA regulating system, sul2, and strAB, and a second part containing tetR, tetA, pecM, and tnpA of Tn1721. Both parts are similar (>99% identity) to a region of plasmid pAPEC-O103-ColBM, but the second part is in the reverse orientation (12). The resistance phenotypes of the TN48 and DH10B/pTN48 strains were consistent with the antibiotic resistance genes identified. Two kinds of macrolide resistance-mediating genes were also identified, mphA, a gene that is commonly present in Enterobacteriaceae, and the ermBC genes, which are common in streptococci but rarely isolated in E. coli (22).Open in a separate windowFIG. 2.Schematic representation of the antibiotic resistance region of plasmid pTN48. Black arrows represent resistance genes, hatched black arrows represent mobile genetic elements, and white arrows represent the other genes or hypothetical-protein-encoding genes. Truncated genes are indicated by a Δ symbol. Vertical bars indicate the inverted 38-bp repeats of Tn2 and Tn1721.The virulence region of approximately 17,000 bp contained a subset of virulence factor coding genes found on ColBM plasmids (12, 13). This region had >99% identity and conserved synteny with the multidrug resistance plasmid pSMS35_130 (9). These included, in order, the colBM gene cluster, ompT, hlyF, mig-14, and sitABCD. To assess the role of these plasmid-borne virulence genes, we tested the extraintestinal virulence of TN48 and DH10B/pTN48 in a mouse model of septicemia as described in reference 10. The TN48 strain was highly virulent, as it killed all 10 of the inoculated mice, as did highly virulent control strain CFT073 (10), whereas strain DH10B/pTN48 was not virulent, as it did not kill any of the 10 mice inoculated, as did the two commensal derived K-12 strains MG1655 (10) and DH10B. This indicates that the plasmid by itself is not able to transform, in our model, an avirulent strain into a virulent strain.The replication-and-maintenance region contained a complete transfer locus of 33,264 bp composed of 24 tra genes, 9 trb genes, artA, and finO. This region was similar in a conserved synteny (98 to 99% identity) to pAPEC-O1-ColBM (11), pSMS35_130 (9), and pIP1206 (21). However, the traV gene, implicated in pore construction, was truncated at the 5′ end by the insertion of IS629. This truncation could putatively impair conjugation efficiency, which is consistent with the observation that pTN48 was not transferable by conjugation in vitro (8). In addition, pTN48 carried several plasmid maintenance and partitioning modules (srnB, pemI pemK, hok mok sok, parB, and sopAB), ensuring stable plasmid inheritance. Actually, the E. coli TN48 and DH10B/pTN48 strains were not cured of the plasmid by sodium dodecyl sulfate (20) or novobiocin (15) treatment or after 200 generations in batch cultures without antibiotic pressure.Plasmid pTN48 has several replicons. A first 17,253-bp region contains two copies of repFII (a and b) separated by an arcABCD gene cluster encoding proteins involved in the arginine deiminase pathway (1); this region was 99% identical to the FII region of pIP1206 (21). In addition, repFIB was located downstream of the ompT hlyF mig-14 virulence region.To assess the phylogenetic history of the pTN48 backbone, a tree was built as described in reference 12, by using the concatenated gene sequences of conserved regions, traM, traX, finO, repA1 (FII), and repA (FIB), from 18 E. coli plasmids of the FIIA-FIB replicon type. The phylogenetic tree generated by the unweighted-pair group method using average linkages (UPGMA) showed that pTN48 did not cluster with the plasmids that share its virulence region, pAPEC and related plasmids (12), which include pS88, a plasmid of E. coli neonatal meningitis (20), and pSMS35 (9), but clustered with plasmids lacking this region, such as pIP1206 (21) (Fig. ​(Fig.3).3). This suggests that acquisition of the virulence region can occur on FIIA-FIB plasmids with distinct phylogenetic backgrounds.Open in a separate windowFIG. 3.Phylogenetic tree of 18 FIIA-FIB E. coli backbone plasmids reconstructed from the concatenated DNA sequences of five genes (repA1, repA, traM, traX, and finO) by the UPGMA. Bootstrap values exceeding 70% are indicated at the nodes. The DNA sequences of plasmids p1658/97 (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF550679.1","term_id":"28629230","term_text":"AF550679.1"}}AF550679.1), pAPEC-O2-ColV ({"type":"entrez-nucleotide","attrs":{"text":"AY545598.5","term_id":"83743321","term_text":"AY545598.5"}}AY545598.5), pVM01 ({"type":"entrez-nucleotide","attrs":{"text":"EU330199.1","term_id":"168830962","term_text":"EU330199.1"}}EU330199.1), pSMS35_130 ({"type":"entrez-nucleotide","attrs":{"text":"CP000971.1","term_id":"170522036","term_text":"CP000971.1"}}CP000971.1), pECOS88 ({"type":"entrez-nucleotide","attrs":{"text":"CU928146.1","term_id":"218349681","term_text":"CU928146.1"}}CU928146.1), pAPEC-O103-ColBM ({"type":"entrez-nucleotide","attrs":{"text":"CP001232.1","term_id":"215252832","term_text":"CP001232.1"}}CP001232.1), pAPEC-1 ({"type":"entrez-nucleotide","attrs":{"text":"CP000836.1","term_id":"221589217","term_text":"CP000836.1"}}CP000836.1), pUTI89 ({"type":"entrez-nucleotide","attrs":{"text":"CP000244.1","term_id":"91075696","term_text":"CP000244.1"}}CP000244.1), pMAR2 ({"type":"entrez-nucleotide","attrs":{"text":"FM180569.1","term_id":"215267788","term_text":"FM180569.1"}}FM180569.1), pVir68 ({"type":"entrez-nucleotide","attrs":{"text":"CP001162.1","term_id":"253721152","term_text":"CP001162.1"}}CP001162.1), pO157 DNA ({"type":"entrez-nucleotide","attrs":{"text":"AB011549.2","term_id":"4589740","term_text":"AB011549.2"}}AB011549.2), pEC14_114 ({"type":"entrez-nucleotide","attrs":{"text":"GQ398086.1","term_id":"256275461","term_text":"GQ398086.1"}}GQ398086.1), pO86A1 DNA ({"type":"entrez-nucleotide","attrs":{"text":"AB255435.1","term_id":"115500638","term_text":"AB255435.1"}}AB255435.1), pECSF1 DNA ({"type":"entrez-nucleotide","attrs":{"text":"AP009379.1","term_id":"281181549","term_text":"AP009379.1"}}AP009379.1), p1ESCUM ({"type":"entrez-nucleotide","attrs":{"text":"CU928148.1","term_id":"218349957","term_text":"CU928148.1"}}CU928148.1), pIP1206 ({"type":"entrez-nucleotide","attrs":{"text":"AM886293.1","term_id":"172051323","term_text":"AM886293.1"}}AM886293.1), and pAPEC-O1-ColBM ({"type":"entrez-nucleotide","attrs":{"text":"DQ381420.1","term_id":"88770133","term_text":"DQ381420.1"}}DQ381420.1) were extracted from GenBank (http://www.ncbi.nlm.nih.gov/GenBank/), but that of plasmid pTN48 (in bold) was not. The presence of virulence genes is indicated at the right by plus signs.In conclusion, pTN48 is a mosaic of antibiotic resistance, virulence, and addiction system modules which appeared to have evolved through stepwise events of integration and/or recombination of DNA modules from various virulence or resistance plasmids. This suggests a high rate of gene flow between bacteria harboring these public-health-threatening plasmids. Yet, similar to the CTX-M-15-encoding F plasmids in O25b-ST131 strains described by Woodford et al. (27), the presence of numerous addiction systems in pTN48 should contribute to plasmid maintenance and therefore multidrug resistance and CTX-M enzyme dissemination (17).     

Structure,Activity, and Inhibition of the Carboxyltransferase β-Subunit of Acetyl Coenzyme A Carboxylase (AccD6) from Mycobacterium tuberculosis

In Mycobacterium tuberculosis, the carboxylation of acetyl coenzyme A (acetyl-CoA) to produce malonyl-CoA, a building block in long-chain fatty acid biosynthesis, is catalyzed by two enzymes working sequentially: a biotin carboxylase (AccA) and a carboxyltransferase (AccD). While the exact roles of the three different biotin carboxylases (AccA1 to -3) and the six carboxyltransferases (AccD1 to -6) in M. tuberculosis are still not clear, AccD6 in complex with AccA3 can synthesize malonyl-CoA from acetyl-CoA. A series of 10 herbicides that target plant acetyl-CoA carboxylases (ACC) were tested for inhibition of AccD6 and for whole-cell activity against M. tuberculosis. From the tested herbicides, haloxyfop, an arylophenoxypropionate, showed in vitro inhibition of M. tuberculosis AccD6, with a 50% inhibitory concentration (IC₅₀) of 21.4 ± 1 μM. Here, we report the crystal structures of M. tuberculosis AccD6 in the apo form (3.0 Å) and in complex with haloxyfop-R (2.3 Å). The structure of M. tuberculosis AccD6 in complex with haloxyfop-R shows two molecules of the inhibitor bound on each AccD6 subunit. These results indicate the potential for developing novel therapeutics for tuberculosis based on herbicides with low human toxicity.     

Ceftazidime-Avibactam Activity Tested against Enterobacteriaceae Isolates from U.S. Hospitals (2011 to 2013) and Characterization of β-Lactamase-Producing Strains

Ceftazidime-avibactam (MIC_50/90, 0.12/0.25 μg/ml) inhibited 99.9% (20,698/20,709) of Enterobacteriaceae isolates at ≤8 μg/ml. This compound was active against resistant subsets, including ceftazidime-nonsusceptible Enterobacter cloacae (MIC_50/90, 0.25/0.5 μg/ml) and extended-spectrum β-lactamase (ESBL) phenotype isolates. An ESBL phenotype was noted among 12.4% (1,696/13,692 isolates from targeted species) of the isolates, including 776 Escherichia coli (12.0% for this species; MIC_50/90, 0.12/0.25 μg/ml), 721 Klebsiella pneumoniae (16.3%; MIC_50/90, 0.12/0.25 μg/ml), 119 Klebsiella oxytoca (10.3%; MIC_50/90, 0.06/0.25 μg/ml), and 80 Proteus mirabilis (4.9%; MIC_50/90, 0.06/0.12 μg/ml) isolates. The most common enzymes detected among ESBL phenotype isolates from 2013 (n = 743) screened using a microarray-based assay were CTX-M-15-like (n = 307), KPC (n = 120), SHV ESBLs (n = 118), and CTX-M-14-like (n = 110). KPC producers were highly resistant to comparators, and ceftazidime-avibactam (MIC_50/90, 0.5/2 μg/ml) and tigecycline (MIC_50/90, 0.5/1 μg/ml; 98.3% susceptible) were the most active agents against these strains. Meropenem (MIC_50/90, ≤0.06/≤0.06 μg/ml) and ceftazidime-avibactam (MIC_50/90, 0.12/0.25 μg/ml) were active against CTX-M-producing isolates. Other enzymes were also observed, and ceftazidime-avibactam displayed good activity against the isolates producing less common enzymes. Among 11 isolates displaying ceftazidime-avibactam MIC values of >8 μg/ml, three were K. pneumoniae strains producing metallo-β-lactamases (all ceftazidime-avibactam MICs, >32 μg/ml), with two NDM-1 producers and one K. pneumoniae strain carrying the bla_KPC-2 and bla_VIM-4 genes. Therapeutic options for isolates producing β-lactamases may be limited, and ceftazidime-avibactam, which displayed good activity against strains, including those producing KPC enzymes, merits further study in infections where such organisms occur.     

The Active Site of ICP47, a Herpes Simplex Virus–encoded Inhibitor of the Major Histocompatibility Complex (MHC)-encoded Peptide Transporter Associated with Antigen Processing (TAP), Maps to the NH2-terminal 35 Residues

The herpes simplex virus (HSV) immediate early protein ICP47 inhibits the transporter associated with antigen processing (TAP)-dependent peptide translocation. As a consequence, empty major histocompatibility complex (MHC) class I molecules are retained in the endoplasmic reticulum and recognition of HSV-infected cells by cytotoxic T lymphocytes is abolished. We chemically synthesized full-length ICP47 (sICP47) and show that sICP47 inhibits TAP-dependent peptide translocation in human cells. Its biological activity is indistinguishable from that of recombinant ICP47 (rICP47). By using synthetic peptides, we mapped the core sequence of ICP47 minimally required for TAP inhibition to residues 2–35. This segment is located within the region of the molecule conserved between ICP47 from HSV-1 and HSV-2. Through alanine scanning substitution we identified three segments within this region that are critical for the ability to inhibit TAP function. The interaction of ICP47 with TAP is unlikely to mimic precisely that of the transported peptides, as deduced from differential labeling of the TAP1 and TAP2 subunits using sICP47 fragments with chemical cross-linkers.The MHC-encoded transporter associated with antigen processing (TAP)¹ connects the cytosol with the lumen of the endoplasmic reticulum (ER) to allow loading of MHC class I molecules with cytosolic peptides for presentation to CTL (1–3). This MHC class I–restricted pathway is critical for elimination of most virus infections. TAP, a key component of this pathway, is blocked specifically by the herpes simplex virus (HSV) protein ICP47, a blockade that allows escape from eradication by CTL (4, 5). TAP is a member of the ATP-binding cassette (ABC) family of transporters, which includes the cystic fibrosis transmembrane conductance regulator (CFTR) and the multidrug resistance transporter (MDR) (6). To date, ICP47 is the only known natural inhibitor of a member of the ABC transporter family. A better understanding of the mode of interaction between ICP47 and TAP is relevant not only for learning more about viral evasion strategies, but could also inspire the design of inhibitors for other members of the ABC transporter family.ICP47 of HSV-1 is an 87–amino acid cytosolic polypeptide, 88 residues if the initiation methionine is included. It binds to the TAP1–TAP2 heterodimer in human but not in mouse cells and prevents transport of peptides through blockade of the peptide binding site of TAP (7, 8). As a consequence, MHC class I molecules fail to be loaded with peptides. The resultant empty class I molecules are retained in the ER and presentation of epitopes to CTL is abolished in HSV-infected human cells (4, 5).The affinity of the human TAP–ICP47 interaction has been estimated to be around 50 nM (9, 10). The ability of ICP47 to prevent photocross-linking of peptides to TAP indicated that ICP47 prohibited peptide binding to TAP (9). Furthermore, the kinetics of competition between peptide and ICP47 for binding to TAP indicate that ICP47 and peptide may compete for a single binding site (9, 10). While suggestive, these experiments cannot readily distinguish between a conformational distortion of TAP caused by ICP47, or a direct competition for the binding site.Here, we have used chemical synthesis to make fulllength ICP47, as well as NH₂- and COOH-terminally truncated versions and alanine-substituted peptide analogues. We show that the ability of ICP47 to inhibit TAP lies within the NH₂-terminal half of the molecule, which is highly conserved between ICP47 from HSV-1 and HSV-2. We present evidence that the mechanism of interaction of ICP47 with the TAP heterodimer likely differs from that of its peptide substrates.     

Molecular characterization of carbapenem-resistant Klebsiella pneumoniae ST14 and ST512 causing bloodstream infections in ICU and surgery wards of a tertiary university hospital of Verona (northern Italy): co-production of KPC-3, OXA-48, and CTX-M-15 β-lactamases

Klebsiella pneumoniae strain is an important opportunistic pathogen that causes severe nosocomial infections. In the present study a molecular characterization of carbapenem resistant K. pneumoniae, isolated from blood samples of hospitalized patients of Verona University Hospital, was performed. The simultaneous presence of SHV-1/CTX-M-15/KPC-3 and SHV-1/CTX-M-15/OXA-48 serin-β-lactamases was ascertained in the 89% and 11% of K. pneumoniae ST512 and K. pneumoniae ST14, respectively. Molecular characterization of bla genes showed that bla_KPC-3 was found in Tn4401a transposon with the tnpR, tnpA, ISKpn6, and ISKpn7 mobile elements whereas bla_CTX-M-15 was detected downstream ISEcp1 genetic element. A class 1 integron with a gene cassette of 780 bp corresponding to aadA2 gene was identified in 33 K. pneumoniae ST512 isolates.     

Multiresistant Uropathogenic Escherichia coli from a Region in India Where Urinary Tract Infections Are Endemic: Genotypic and Phenotypic Characteristics of Sequence Type 131 Isolates of the CTX-M-15 Extended-Spectrum-β-Lactamase-Producing Lineage

Escherichia coli sequence type 131 (O25b:H4), associated with the CTX-M-15 extended-spectrum beta-lactamases (ESBLs) and linked predominantly to the community-onset antimicrobial-resistant infections, has globally emerged as a public health concern. However, scant attention is given to the understanding of the molecular epidemiology of these strains in high-burden countries such as India. Of the 100 clinical E. coli isolates obtained by us from a setting where urinary tract infections are endemic, 16 ST131 E. coli isolates were identified by multilocus sequence typing (MLST). Further, genotyping and phenotyping methods were employed to characterize their virulence and drug resistance patterns. All the 16 ST131 isolates harbored the CTX-M-15 gene, and half of them also carried TEM-1; 11 of these were positive for bla_OXA groups 1 and 12 for aac(6′)-Ib-cr. At least 12 isolates were refractory to four non-beta-lactam antibiotics: ciprofloxacin, gentamicin, sulfamethoxazole-trimethoprim, and tetracycline. Nine isolates carried the class 1 integron. Plasmid analysis indicated a large pool of up to six plasmids per strain with a mean of approximately three plasmids. Conjugation and PCR-based replicon typing (PBRT) revealed that the spread of resistance was associated with the FIA incompatibility group of plasmids. Pulsed-field gel electrophoresis (PFGE) and genotyping of the virulence genes showed a low level of diversity among these strains. The association of ESBL-encoding plasmid with virulence was demonstrated in transconjugants by serum assay. None of the 16 ST131 ESBL-producing E. coli strains were known to synthesize carbapenemase enzymes. In conclusion, our study reports a snapshot of the highly virulent/multiresistant clone ST131 of uropathogenic E. coli from India. This study suggests that the ST131 genotypes from this region are clonally evolved and are strongly associated with the CTX-M-15 enzyme, carry a high antibiotic resistance background, and have emerged as an important cause of community-acquired urinary tract infections.     

Molecular epidemiological analysis of Escherichia coli sequence type ST131 (O25:H4) and blaCTX-M-15 among extended-spectrum-β-lactamase-producing E. coli from the United States, 2000 to 2009

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.