Cunningham, Scott A.

Escherichia colibacteremia is caused mainly by sequence type complex 131 (STc131) and two clades within its fluoroquinolone-resistance-associatedH30 subclone,H30R1 andH30Rx. We examined clinical and molecular correlates ofE. colibacteremia in two geographically distinct centers.

Background Kingella kingae is a known cause of osteoarticular infections in children younger than 4 years of age, but it is not always recoverable in culture. Molecular methods are increasingly used for diagnosis. Methods To facilitate diagnosis of K. kingae septic arthritis, we developed a real-time polymerase chain reaction (PCR) assay for the detection of K. kingae that targets the repeat-in-toxin gene (rtxB). Results We present three pediatric patients with K. kingae septic arthritis at our institution who were diagnosed using the real-time PCR assay. All underwent arthrotomy with irrigation and debridement and were symptom-free after 3 weeks of therapy with β-lactam antibiotics. Cultures of synovial fluid or tissue grew K. kingae in two of three; K. kingae real-time PCR was positive in all three patients. In addition, 11 cases of K. kingae osteoarticular infection were diagnosed through Mayo Medical Laboratories using this assay. The limit of detection of the real-time PCR assay was 73.7 colony-forming unit (CFU)/µL for tissue and 1.3 CFU/µL for synovial fluid. Conclusions PCR-based detection methods are faster and more sensitive than conventional culture-based methods for the diagnosis of K. kingae osteoarticular infections in children.

ABSTRACT Eighty Gram-negative bacilli (54 Enterobacteriaceae and 26 nonfermenting Gram-negative bacilli) obtained from multiple institutions in the United States were distributed in a blinded manner to seven testing laboratories to compare their performance of a test for detection of carbapenemase production, the Carba NP test. The Carba NP test was performed by all laboratories, following the Clinical and Laboratory Standards Institute (CLSI) procedure. Site-versus-site comparisons demonstrated a high level of consistency for the Carba NP assay, with just 3/21 site comparisons yielding a difference in sensitivity ( P < 0.05). Previously described limitations with bla OXA-48-like carbapenemases and bla OXA carbapenemases associated with Acinetobacter baumannii were noted. Based on these data, we demonstrate that the Carba NP test, when implemented with the standardized CLSI methodology, provides reproducible results across multiple sites for detection of carbapenemases.

ABSTRACT Whole-genome sequencing (WGS) can provide excellent resolution in global and local epidemiological investigations of Staphylococcus aureus outbreaks. A variety of sequencing approaches and analytical tools have been used; it is not clear which is ideal. We compared two WGS strategies and two analytical approaches to the standard method of SmaI restriction digestion pulsed-field gel electrophoresis (PFGE) for typing S. aureus . Forty-two S. aureus isolates from three outbreaks and 12 reference isolates were studied. Near-complete genomes, assembled de novo with paired-end and long-mate-pair (8 kb) libraries were first assembled and analyzed utilizing an in-house assembly and analytical informatics pipeline. In addition, paired-end data were assembled and analyzed using a commercial software package. Single nucleotide variant (SNP) analysis was performed using the in-house pipeline. Two assembly strategies were used to generate core genome multilocus sequence typing (cgMLST) data. First, the near-complete genome data generated with the in-house pipeline were imported into the commercial software and used to perform cgMLST analysis. Second, the commercial software was used to assemble paired-end data, and resolved assemblies were used to perform cgMLST. Similar isolate clustering was observed using SNP calling and cgMLST, regardless of data assembly strategy. All methods provided more discrimination between outbreaks than did PFGE. Overall, all of the evaluated WGS strategies yielded statistically similar results for S. aureus typing.

Tropheryma whipplei, the agent of Whipple disease, causes a rare bacterial disease that may be fatal if not treated. The classical form of the disease includes diarrhoea, weight loss, arthritis, endocarditis and neurological manifestations. Genotyping studies done in Europe, Africa and Asia showed high genetic diversity with no correlation between genotypes and clinical features, but contributed to a better understanding of the epidemiology of the disease. More than 70 genotypes have been described. No similar assessment of T. whipplei in the USA and the Caribbean has been performed. In this study, we describe genetic analysis of DNA from histopathological samples obtained from 30 patients from the Americas with Whipple disease and compare the genotypes with those previously identified. Complete genotypes were obtained from 18 patients (60%). Only 4 genotypes were previously described, and 14 were newly reported, confirming the diversity of T. whipplei strains.

ABSTRACT Failure to eradicate Helicobacter pylori infection is often a result of antimicrobial resistance, which for clarithromycin is typically mediated by specific point mutations in the 23S rRNA gene. The purpose of this study was to define current patterns of antimicrobial susceptibility in H. pylori isolates derived primarily from the United States and to survey them for the presence of point mutations in the 23S rRNA gene and assess the ability of these mutations to predict phenotypic clarithromycin susceptibility. Antimicrobial susceptibility testing was performed using agar dilution on 413 H. pylori isolates submitted to Mayo Medical Laboratories for susceptibility testing. For a subset of these isolates, a 150-bp segment of the 23S rRNA gene was sequenced. A total of 1,970 MICs were reported over the 4-year study period. The rate of clarithromycin resistance was high (70.4%), and elevated MICs were frequently observed for metronidazole (82.4% of isolates had an MIC of >8 μg/ml) and ciprofloxacin (53.5% of isolates had an MIC of >1 μg/ml). A total of 111 archived H. pylori isolates underwent 23S rRNA gene sequencing; we found 95% concordance between genotypes and phenotypes ( P = 0.9802). Resistance to clarithromycin was most commonly due to an A2143G mutation (82%), followed by A2142G (14%) and A2142C (4%) mutations. Clinical H. pylori isolates derived primarily from the United States demonstrated a high rate of clarithromycin resistance and elevated metronidazole and ciprofloxacin MICs. The relative distribution of point mutations at positions 2143 and 2142 in the 23S rRNA gene in clarithromycin-resistant H. pylori was similar to that reported from other parts of the world; these mutations predict phenotypic resistance to clarithromycin.

ABSTRACT Ureaplasma urealyticum and Ureaplasma parvum are pathogens involved in urogenital tract and intrauterine infections and also in systemic diseases in newborns and immunosuppressed patients. There is limited information on the antimicrobial susceptibility and clonality of these species. In this study, we report the susceptibility of 250 contemporary isolates of Ureaplasma (202 U. parvum and 48 U. urealyticum isolates) recovered at Mayo Clinic, Rochester, MN. MICs of doxycycline, azithromycin, ciprofloxacin, tetracycline, erythromycin, and levofloxacin were determined by broth microdilution, with MICS of the last three interpreted according to CLSI guidelines. Levofloxacin resistance was found in 6.4% and 5.2% of U. parvum and U. urealyticum isolates, respectively, while 27.2% and 68.8% of isolates, respectively, showed ciprofloxacin MICs of ≥4 μg/ml. The resistance mechanism of levofloxacin-resistant isolates was due to mutations in parC , with the Ser83Leu substitution being most frequent, followed by Glu87Lys. No macrolide resistance was found among the 250 isolates studied; a single U. parvum isolate was tetracycline resistant. tet (M) was found in 10 U. parvum isolates, including the single tetracycline-resistant isolate, as well as in 9 isolates which had low tetracycline and doxycycline MICs. Multilocus sequence typing (MLST) performed on a selection of 46 isolates showed high diversity within the clinical Ureaplasma isolates studied, regardless of antimicrobial susceptibility. The present work extends previous knowledge regarding susceptibility to antimicrobial agents, resistance mechanisms, and clonality of Ureaplasma species in the United States.