Pandemic Uropathogenic Fluoroquinolone-resistant Escherichia coli Have Enhanced Ability to Persist in the Gut and Cause Bacteriuria in Healthy Women.

We report that fluoroquinolone-resistant Escherichia coli are found in feces of 8.8% of healthy women, with most bacteria belonging to pandemic multidrug-resistant ST131-H30R or ST1193 clonal groups. Moreover, these highly uropathogenic clonal groups demonstrate an especially prolonged gut persistence and high rate of bacteriuria without documented urinary tract infection.

The Uropathogenic Escherichia coli Subclone Sequence Type 131-H30 Is Responsible for Most Antibiotic Prescription Errors at an Urgent Care Clinic.

BACKGROUND: The pandemic spread of antibiotic resistance increases the likelihood of ineffective empirical therapy. The recently emerged fluoroquinolone-resistant Escherichia coli sequence type (ST) 131-H30R subclone (H30) is a leading cause of multidrug-resistant urinary tract infection (UTI) and bloodstream infection worldwide. METHODS: We studied the relative impact of H30 on the likelihood that bacteria isolated from urine of urgent care patients would be resistant to the empirically prescribed antibiotic regimen for UTI. RESULTS: Of 750 urinalysis-positive urine samples from urgent care patients with suspected UTI, 306 (41%) yielded E. coli, from 35 different clonal groups (clonotypes). H30 predominated (14% prevalence overall), especially among older patients (age ≥70 years: 26%) and those with diabetes (43%) or urinary catheterization (60%). Resistance to the empirically selected antibiotic regimen occurred in 16% (40/246) of patients overall, 28% (20/71) of older patients, 30% (8/27) of patients with diabetes, 60% (3/5) of catheterized patients, and 71% (22/30) of those with H30. H30's contribution to such mismatched antibiotic selection was 55% overall, 70% among older patients, and 100% among patients with diabetes or a urinary catheter. Among patients with ≥2 of these factors (older age, diabetes, or urinary catheter), 24% of all urinalysis-positive urine samples yielded H30, with a 92% likelihood of resistance to the selected empirical therapy. CONCLUSIONS: The multidrug-resistant H30 subclone of E. coli ST131 is responsible for the great majority of mismatched empirical antibiotic prescriptions for suspected UTI at an urgent care clinic among patients ≥70 years old or with diabetes or urinary catheterization.

Rapid and Extensive Expansion in the United States of a New Multidrug-resistant Escherichia coli Clonal Group, Sequence Type 1193.

We describe the rapid and ongoing emergence across multiple US cities of a new multidrug-resistant Escherichia coli clone-sequence type (ST) 1193-resistant to fluoroquinolones (100%), trimethoprim-sulfamethoxazole (55%), and tetracycline (53%). ST1193 is associated with younger adults (age <40 years) and currently comprises a quarter of fluoroquinolone-resistant clinical E. coli urine isolates.

Bacterial clonal diagnostics as a tool for evidence-based empiric antibiotic selection.

Despite the known clonal distribution of antibiotic resistance in many bacteria, empiric (pre-culture) antibiotic selection still relies heavily on species-level cumulative antibiograms, resulting in overuse of broad-spectrum agents and excessive antibiotic/pathogen mismatch. Urinary tract infections (UTIs), which account for a large share of antibiotic use, are caused predominantly by Escherichia coli, a highly clonal pathogen. In an observational clinical cohort study of urgent care patients with suspected UTI, we assessed the potential for E. coli clonal-level antibiograms to improve empiric antibiotic selection. A novel PCR-based clonotyping assay was applied to fresh urine samples to rapidly detect E. coli and the urine strain's clonotype. Based on a database of clonotype-specific antibiograms, the acceptability of various antibiotics for empiric therapy was inferred using a 20%, 10%, and 30% allowed resistance threshold. The test's performance characteristics and possible effects on prescribing were assessed. The rapid test identified E. coli clonotypes directly in patients' urine within 25-35 minutes, with high specificity and sensitivity compared to culture. Antibiotic selection based on a clonotype-specific antibiogram could reduce the relative likelihood of antibiotic/pathogen mismatch by ≥ 60%. Compared to observed prescribing patterns, clonal diagnostics-guided antibiotic selection could safely double the use of trimethoprim/sulfamethoxazole and minimize fluoroquinolone use. In summary, a rapid clonotyping test showed promise for improving empiric antibiotic prescribing for E. coli UTI, including reversing preferential use of fluoroquinolones over trimethoprim/sulfamethoxazole. The clonal diagnostics approach merges epidemiologic surveillance, antimicrobial stewardship, and molecular diagnostics to bring evidence-based medicine directly to the point of care.

The Pandemic H30 Subclone of Escherichia coli Sequence Type 131 Is Associated With Persistent Infections and Adverse Outcomes Independent From Its Multidrug Resistance and Associations With Compromised Hosts.

BACKGROUND: The H30 subclone within Escherichia coli sequence type 131 (ST131-H30) has emerged rapidly to become the leading antibiotic-resistant E. coli strain. Hypervirulence, multidrug resistance, and opportunism have been proposed as explanations for its epidemic success. METHODS: We assessed 1133 consecutive unique E. coli clinical isolates from 5 medical centers (2010-2011) for H30 genotype, which we compared with epidemiological and clinical data extracted from medical records by blinded reviewers. Using univariable and multivariable logistic regression analysis, we explored associations of H30 with underlying host characteristics, clinical presentations, management, and outcomes, adjusting for host characteristics. RESULTS: The H30 (n = 107) isolates were associated with hosts who were older, male, locally and systemically compromised, and healthcare and antibiotic exposed. With multivariable adjustment for host factors, H30 lost its numerous significant univariable associations with initial clinical presentation, but remained strongly associated with clinical persistence (odds ratio [OR], 3.47; 95% confidence interval [CI], 1.89-6.37), microbiological persistence (OR, 4.46; 95% CI, 2.38-8.38), subsequent hospital admission (OR, 2.68; 95% CI, 1.35-5.33), and subsequent new infection (OR, 1.73; 95% CI, 1.01-3.00). These host-adjusted associations remained strong even with added adjustment for resistance to the initially prescribed antibiotics, and the adverse outcome associations (subsequent hospital admission, new infection) were independent of clinical and microbiological persistence. CONCLUSIONS: In addition to targeting compromised hosts and resisting multiple antibiotics, H30 isolates may have an intrinsic ability to cause highly persistent infections and later adverse outcomes. The basis for these host- and resistance-independent associations is unclear, but they should be considered when managing patients with H30 infections.

A Novel 7-Single Nucleotide Polymorphism-Based Clonotyping Test Allows Rapid Prediction of Antimicrobial Susceptibility of Extraintestinal Escherichia coli Directly From Urine Specimens.

Background. Escherichia coli is a highly clonal pathogen. Extraintestinal isolates belong to a limited number of genetically related groups, which often exhibit characteristic antimicrobial resistance profiles. Methods. We developed a rapid clonotyping method for extraintestinal E coli based on detection of the presence or absence of 7 single nucleotide polymorphisms (SNPs) within 2 genes (fumC and fimH). A reference set of 2559 E coli isolates, primarily of urinary origin, was used to predict the resolving power of the 7-SNP-based typing method, and 582 representative strains from this set were used to evaluate test robustness. Results. Fifty-four unique SNP combinations ("septatypes") were identified in the reference strains. These septatypes yielded a clonal group resolution power on par with that of traditional multilocus sequence typing. In 72% of isolates, septatype identity predicted sequence type identity with at least 90% (mean, 97%) accuracy. Most septatypes exhibited highly distinctive antimicrobial susceptibility profiles. The 7-SNP-based test could be performed with high specificity and sensitivity using single or multiplex conventional polymerase chain reaction (PCR) and quantitative PCR. In the latter format, E coli presence and septatype identity were determined directly in urine specimens within 45 minutes with bacterial loads as low as 10(2) colony-forming units/mL and, at clinically significant bacterial loads, with 100% sensitivity and specificity. Conclusions. 7-SNP-based typing of E coli can be used for both epidemiological studies and clinical diagnostics, which could greatly improve the empirical selection of antimicrobial therapy.

Chronic opioid therapy urine drug testing in primary care: prevalence and predictors of aberrant results.

BACKGROUND: Urine drug tests (UDTs) are recommended for patients on chronic opioid therapy (COT). Knowledge of the risk factors for aberrant UDT results could help optimize their use. OBJECTIVE: To identify primary care COT patient and opioid regimen characteristics associated with aberrant UDT results. DESIGN: Population-based observational. SAMPLE: 5,420 UDTs for Group Health integrated group practice COT patients. MEASURES: Group Health database measures of patient demographics, medical history, COT characteristics, and UDT results. RESULTS: Thirty percent of UDTs had aberrant results, including prescribed opioid non-detection (12.3%), tetrahydrocannabinol (THC; 11.2%), non-prescribed opioid (5.3%), illicit drug (excluding THC; 0.6%), non-prescribed benzodiazepine (1.7%), and dilute (4.8%). Adjusted odds ratios (95% CI) of any aberrant result were higher for males than females (1.24 [1.07, 1.43]), patients with versus without prior substance use disorder diagnoses (1.42 [1.17, 1.72]), and current smokers versus non-smokers (1.50 [1.30, 1.73]). Odds ratios were lower for patients aged 45-64 (0.77 [0.65, 0.92]) and 65+ (0.40 [0.32, 0.50]) versus patients aged 20-44 and for patients on long-acting opioids only (0.72 [0.55, 0.95]) or long-acting plus short-acting (0.67 [0.54, 0.83]) versus short-acting only. Adjusted odds of prescribed opioid non-detection were lower for patients aged 45-64 (0.79 [0.63, 0.998]) and 65+ (0.44 [0.32, 0.59]) versus patients aged 20-44, for those on 40-<120 mg daily morphine-equivalent dose (0.52 [0.39, 0.70]) or 120+ mg (0.22 [0.11, 0.43]) versus <40 mg, and for patients on long-acting (0.35 [0.21, 0.57]) or long-acting plus short-acting (0.35 [0.24, 0.50]) opioids (versus short-acting only); and odds ratios were higher for patients with versus without prior diagnoses of substance use disorder (1.70 [1.31, 2.20]). CONCLUSIONS: In this primary care setting, results were aberrant for 30% of UDTs of COT patients, largely because of prescribed opioid non-detection and THC. Aberrant results of almost all types were more likely among patients under the age of 45. Other risk factors varied across aberrancies, but commonly included current smoking and prior substance use disorder diagnosis.

Chronic opioid therapy risk reduction initiative: impact on urine drug testing rates and results.

BACKGROUND: In response to epidemic levels of prescription opioid overdose, abuse, and diversion, routine urine drug tests (UDTs) are recommended for patients receiving chronic opioid therapy (COT) for chronic pain. However, UDT ordering for COT patients is inconsistent in primary care, and little is known about how to increase UDT ordering or the impact of increased testing on rates of aberrant results. OBJECTIVE: To compare rates and results of UDTs for COT patients before versus after implementation of an opioid risk reduction initiative in a large healthcare system. DESIGN: Pre-post observational study. PATIENTS: Group Health patients on COT October 2008-September 2009 (N = 4,821), October 2009-September 2010 (N = 5,081), and October 2010-September 2011 (N = 5,498). INTERVENTION: Multi-faceted opioid risk reduction initiative. MAIN MEASURES: Annual rates of UDTs and UDT results. KEY RESULTS: Half of COT patients received at least one UDT in the year after the initiative was implemented, compared to only 7 % 2 years prior. The adjusted odds of COT patients having at least one UDT in the first year of the opioid initiative were almost 16 times (adjusted OR = 15.79; 95 % CI: 13.96-17.87) those 2 years prior. The annual rate of UDT detection of marijuana and illicit drugs did not change (12.6 % after initiative implementation), and largely reflected marijuana use (detected in 11.1 % of all UDTs in the year after initiative implementation). In the year after initiative implementation, 10.7 % of UDTs were negative for opioids. CONCLUSIONS: The initiative appeared to dramatically increase urine drug testing of COT patients in the healthcare system without impacting rates of aberrant results. The large majority of aberrant results reflected marijuana use or absence of opioids in the urine. The utility of increased urine drug testing for COT patient safety and prevention of diversion remains uncertain.

The epidemic of extended-spectrum-ß-lactamase-producing Escherichia coli ST131 is driven by a single highly pathogenic subclone, H30-Rx.

UNLABELLED: The Escherichia coli sequence type 131 (ST131) clone is notorious for extraintestinal infections, fluoroquinolone resistance, and extended-spectrum beta-lactamase (ESBL) production, attributable to a CTX-M-15-encoding mobile element. Here, we applied pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing to reconstruct the evolutionary history of the ST131 clone. PFGE-based cluster analyses suggested that both fluoroquinolone resistance and ESBL production had been acquired by multiple ST131 sublineages through independent genetic events. In contrast, the more robust whole-genome-sequence-based phylogenomic analysis revealed that fluoroquinolone resistance was confined almost entirely to a single, rapidly expanding ST131 subclone, designated H30-R. Strikingly, 91% of the CTX-M-15-producing isolates also belonged to a single, well-defined clade nested within H30-R, which was named H30-Rx due to its more extensive resistance. Despite its tight clonal relationship with H30Rx, the CTX-M-15 mobile element was inserted variably in plasmid and chromosomal locations within the H30-Rx genome. Screening of a large collection of recent clinical E. coli isolates both confirmed the global clonal expansion of H30-Rx and revealed its disproportionate association with sepsis (relative risk, 7.5; P < 0.001). Together, these results suggest that the high prevalence of CTX-M-15 production among ST131 isolates is due primarily to the expansion of a single, highly virulent subclone, H30-Rx. IMPORTANCE: We applied an advanced genomic approach to study the recent evolutionary history of one of the most important Escherichia coli strains in circulation today. This strain, called sequence type 131 (ST131), causes multidrug-resistant bladder, kidney, and bloodstream infections around the world. The rising prevalence of antibiotic resistance in E. coli is making these infections more difficult to treat and is leading to increased mortality. Past studies suggested that many different ST131 strains gained resistance to extended-spectrum cephalosporins independently. In contrast, our research indicates that most extended-spectrum-cephalosporin-resistant ST131 strains belong to a single highly pathogenic subclone, called H30-Rx. The clonal nature of H30-Rx may provide opportunities for vaccine or transmission prevention-based control strategies, which could gain importance as H30-Rx and other extraintestinal pathogenic E. coli subclones become resistant to our best antibiotics.

Predictive diagnostics for Escherichia coli infections based on the clonal association of antimicrobial resistance and clinical outcome.

The ability to identify bacterial pathogens at the subspecies level in clinical diagnostics is currently limited. We investigated whether splitting Escherichia coli species into clonal groups (clonotypes) predicts antimicrobial susceptibility or clinical outcome. A total of 1,679 extraintestinal E. coli isolates (collected from 2010 to 2012) were collected from one German and 5 U.S. clinical microbiology laboratories. Clonotype identity was determined by fumC and fimH (CH) sequencing. The associations of clonotype with antimicrobial susceptibility and clinical variables were evaluated. CH typing divided the isolates into >200 CH clonotypes, with 93% of the isolates belonging to clonotypes with ≥ 2 isolates. Antimicrobial susceptibility varied substantially among clonotypes but was consistent across different locations. Clonotype-guided antimicrobial selection significantly reduced "drug-bug" mismatch compared to that which occurs with the use of conventional empirical therapy. With trimethoprim-sulfamethoxazole and fluoroquinolones, the drug-bug mismatch was predicted to decrease 62% and 78%, respectively. Recurrent or persistent urinary tract infection and clinical sepsis were significantly correlated with specific clonotypes, especially with CH40-30 (also known as H30), a recently described clonotype within sequence type 131 (ST131). We were able to clonotype directly from patient urine samples within 1 to 3 h of obtaining the specimen. In E. coli, subspecies-level identification by clonotyping can be used to significantly improve empirical predictions of antimicrobial susceptibility and clinical outcomes in a timely manner.

Abrupt emergence of a single dominant multidrug-resistant strain of Escherichia coli.

BACKGROUND: Fluoroquinolone-resistant Escherichia coli are increasingly prevalent. Their clonal origins--potentially critical for control efforts--remain undefined. METHODS: Antimicrobial resistance profiles and fine clonal structure were determined for 236 diverse-source historical (1967-2009) E. coli isolates representing sequence type ST131 and 853 recent (2010-2011) consecutive E. coli isolates from 5 clinical laboratories in Seattle, Washington, and Minneapolis, Minnesota. Clonal structure was resolved based on fimH sequence (fimbrial adhesin gene: H subclone assignments), multilocus sequence typing, gyrA and parC sequence (fluoroquinolone resistance-determining loci), and pulsed-field gel electrophoresis. RESULTS: Of the recent fluoroquinolone-resistant clinical isolates, 52% represented a single ST131 subclonal lineage, H30, which expanded abruptly after 2000. This subclone had a unique and conserved gyrA/parC allele combination, supporting its tight clonality. Unlike other ST131 subclones, H30 was significantly associated with fluoroquinolone resistance and was the most prevalent subclone among current E. coli clinical isolates, overall (10.4%) and within every resistance category (11%-52%). CONCLUSIONS: Most current fluoroquinolone-resistant E. coli clinical isolates, and the largest share of multidrug-resistant isolates, represent a highly clonal subgroup that likely originated from a single rapidly expanded and disseminated ST131 strain. Focused attention to this strain will be required to control the fluoroquinolone and multidrug-resistant E. coli epidemic.

High-resolution two-locus clonal typing of extraintestinal pathogenic Escherichia coli.

Multilocus sequence typing (MLST) is usually based on the sequencing of 5 to 8 housekeeping loci in the bacterial chromosome and has provided detailed descriptions of the population structure of bacterial species important to human health. However, even strains with identical MLST profiles (known as sequence types or STs) may possess distinct genotypes, which enable different eco- or pathotypic lifestyles. Here we describe a two-locus, sequence-based typing scheme for Escherichia coli that utilizes a 489-nucleotide (nt) internal fragment of fimH (encoding the type 1 fimbrial adhesin) and the 469-nt internal fumC fragment used in standard MLST. Based on sequence typing of 191 model commensal and pathogenic isolates plus 853 freshly isolated clinical E. coli strains, this 2-locus approach-which we call CH (fumC/fimH) typing-consistently yielded more haplotypes than standard 7-locus MLST, splitting large STs into multiple clonal subgroups and often distinguishing different within-ST eco- and pathotypes. Furthermore, specific CH profiles corresponded to specific STs, or ST complexes, with 95% accuracy, allowing excellent prediction of MLST-based profiles. Thus, 2-locus CH typing provides a genotyping tool for molecular epidemiology analysis that is more economical than standard 7-locus MLST but has superior clonal discrimination power and, at the same time, corresponds closely to MLST-based clonal groupings.

AKT promotes rRNA synthesis and cooperates with c-MYC to stimulate ribosome biogenesis in cancer.

Precise regulation of ribosome biogenesis is fundamental to maintain normal cell growth and proliferation, and accelerated ribosome biogenesis is associated with malignant transformation. Here, we show that the kinase AKT regulates ribosome biogenesis at multiple levels to promote ribosomal RNA (rRNA) synthesis. Transcription elongation by RNA polymerase I, which synthesizes rRNA, required continuous AKT-dependent signaling, an effect independent of AKT's role in activating the translation-promoting complex mTORC1 (mammalian target of rapamycin complex 1). Sustained inhibition of AKT and mTORC1 cooperated to reduce rRNA synthesis and ribosome biogenesis by additionally limiting RNA polymerase I loading and pre-rRNA processing. In the absence of growth factors, constitutively active AKT increased synthesis of rRNA, ribosome biogenesis, and cell growth. Furthermore, AKT cooperated with the transcription factor c-MYC to synergistically activate rRNA synthesis and ribosome biogenesis, defining a network involving AKT, mTORC1, and c-MYC as a master controller of cell growth. Maximal activation of c-MYC-dependent rRNA synthesis in lymphoma cells required AKT activity. Moreover, inhibition of AKT-dependent rRNA transcription was associated with increased lymphoma cell death by apoptosis. These data indicate that decreased ribosome biogenesis is likely to be a fundamental component of the therapeutic response to AKT inhibitors in cancer.