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 ( fum C / fim H ) 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.

Escherichia coli associated with early-onset sepsis (EOS) have historically been antibiotic-susceptible and K1-encapsulated. In the era of emerging antibiotic resistance, however, the clonal makeup of E coli associated with EOS has not been well characterized. Escherichia coli isolates were collected from 28 cases of EOS and early-onset meningitis (EOM) from April 2008 through December 2009, during a parent study conducted at National Institute of Child Health and Human Development Neonatal Research Network centers from February 2006 through December 2009. Clinical and microbiologic data were collected for the parent study. We applied polymerase chain reaction- and sequence-based molecular techniques to determine clonal, virulence-associated and antibiotic resistance-associated traits of the E coli isolates. Among 28 E coli strains, phylogroup B2 strains predominated (68%), of which more than half were K1-encapsulated (53%). Phylogroup D strains were prominent as well (18%), but none were K1-encapsulated. Across the strain collection, the rate of ampicillin resistance was high (78%). The sole strain resistant to either extended-spectrum cephalosporins or fluoroquinolones represented ST131 H 30-Rx, the multidrug-resistant subclone that has emerged worldwide in the last decade. This strain encoded extended-spectrum β-lactamase CTX-M-15 and carried an IncF plasmid of type F2:A1:B-. In this collection of EOS/EOM-associated E coli isolates, we observed a high rate of ampicillin resistance, a low rate of fluoroquinolone resistance, and no aminoglycoside resistance, with resistance to third-generation cephalosporins appearing in only a single strain, from the worldwide emerging ST131 clone. Ongoing surveillance of antibiotic resistance among EOS isolates is warranted, to ensure that standard empiric regimens remain effective.

Klebsiella pneumoniae causes a range of clinical disease in paediatric patients and is of increasing concern due to growing antibiotic resistance, yet little is known about the relative distribution of commensal and pathogens throughout the population structure of K. pneumoniae. We conducted a prospective, observational study of 92 isolates from Seattle Children’s Hospital, including 49 disease isolates from blood and urine (13 and 36 isolates, respectively) and 43 colonization isolates from stool. Susceptibility to 20 antimicrobials was evaluated using disc diffusion, VITEK 2 and Etest. Strain relatedness was investigated using multilocus sequence typing (MLST). Demographic and clinical characteristics were largely similar between disease and colonization cohorts, with 85.7 and 74.4 % of disease and colonization cohort patients, respectively, having an underlying medical condition; the sole exception was a relative abundance of patients with urologic or renal abnormalities in the disease cohort, consistent with the predominance of urine specimens among the disease isolates. With regard to antibiotic susceptibility properties, no significant differences were noted between the disease and colonization cohorts. Using molecular analysis, 71 unique sequence types (STs) were distinguished, with novel MLST findings evident in both cohorts; 43 (46.7 %) isolates represented novel STs, including 22 with a novel allele sequence. Thirteen STs contained multiple isolates and all seven isolates with resistance to three or more antibiotic classes were within one of four multirepresentative STs. This study demonstrates that nearly half of paediatric Klebsiella isolates represent novel STs, with clustering of multidrug resistance within specific STs. These findings expand our understanding of the intersection of bacterial population structure, human colonization ecology and multidrug resistance in K. pneumoniae.

E. coli was isolated from the Salish Sea (Puget Sound) ecosystem, including samples of marine and fresh water, and wildlife dependent on this environment. E. coli isolates were assessed for phenotypic and genotypic resistance to antibiotics. A total of 305 E. coli isolates was characterized from samples collected from: marine water obtained in four quadrants of the Salish Sea; select locations near beaches; fresh water from streams near marine beaches; and fecal samples from harbor porpoises (Phocoena phocoena), harbor seals (Phoca vitulina), river otters (Lontra canadensis), and English sole (Parophrys vetulus). Isolates were evaluated using antimicrobial susceptibility typing, whole-genome sequencing, fumC, and multilocus sequence typing. Resistance and virulence genes were identified from sequence data. Of the 305 isolates from Salish Sea samples, 20 (6.6%) of the E. coli were intermediate, and 31 (10.2%) were resistant to ≥1 class of antibiotics, with 26.9% of nonsusceptible (resistant and intermediate resistant) E. coli isolates from marine mammals and 70% from river otters. The proportion of nonsusceptible isolates from animals was significantly higher than samples taken from marine water (p < 0.0001). A total of 196 unique STs was identified including 37 extraintestinal pathogenic E. coli (ExPEC)-associated STs [ST10, ST38, ST58, ST69, ST73, ST117, ST131, and ST405]. The study suggests that animals may be potential sentinels for antibiotic-resistant and ExPEC E. coli in the Salish Sea ecosystem.

Providers must balance effective empiric therapy against toxicity risks and collateral damage when selecting antibiotic therapy for patients receiving hematopoietic cell transplant (HCT). Antimicrobial stewardship (AMS) interventions during HCT are often challenging due to concern for undertreating potential infections. In an effort to decrease unnecessary carbapenem exposure for patients undergoing HCT at our pediatric center, we implemented individualized antibiotic plans (IAPs) to provide recommendations for pre-engraftment neutropenia prophylaxis, empiric treatment of febrile neutropenia, and empiric treatment for hemodynamic instability. We compared monthly antibiotic days of therapy adjusted per 1000 patient days (DOT/1000 pt-days) for carbapenems, anti-pseudomonal cephalosporins, and all antibiotics during two 3-year periods immediately before and after the implementation of IAPs to measure the impact of IAP on prescribing behavior. Bloodstream infections (BSI) and Clostridioides difficile (CD) positive test rates were also compared between cohorts. Lastly, providers were surveyed to assess their experience of using IAPs in antibiotic decision-making. Overall antibiotic use decreased after the implementation of IAP (monthly reduction of 19.6 DOT/1000 pt-days, p=0.004) with carbapenems showing a continuing decline after IAP implementation. BSI and CD positivity rates were unchanged. Over 90% of providers found IAPs to be either extremely or very valuable for their practice. Implementation of IAPs in this high-risk HCT population led to reduction in overall antibiotic use without increase in rate of BSI or CD test positivity. The program was well-received by providers.