Short-read whole genome sequencing for determination of antimicrobial resistance mechanisms and capsular serotypes of current invasive Streptococcus agalactiae recovered in the USA.

OBJECTIVES: Our objective was to evaluate and exploit a whole genome sequence (WGS) bioinformatics pipeline for predicting antimicrobial resistance and capsular serotypes from invasive group B streptococci (iGBS). METHODS: For 1975 iGBS recovered during 2015 from CDC's Active Bacterial Core surveillance, we compared pipeline predictions with broth dilution testing. Fifty-six isolates from earlier surveillance were included for testing ß-lactams. Conventional serotyping was compared to WGS-based assignments for 302 isolates. RESULTS: All 28 isolates with reduced susceptibility to ß-lactam antibiotics harboured one of 19 rare PBP2x types. Resistances to erythromycin/clindamycin (808/1975 isolates, 41.0%), erythromycin (235/1975, 11.9%) and lincosamide/streptogramin A/pleuromutilins (56/1975, 2.8%) were predicted by the presence of erm-methylase, mef and lsa determinants, respectively (41 of 56 lsa gene-positive isolates also contained lnu, erm and/or mef genes). Presence of both erm and lsa determinants (25 isolates) predicted non-susceptibility to quinupristin/dalfopristin. Most isolates (1680/1975, 85.1%) were tet gene-positive, although 41/1565 (2.6%) tetM-positive isolates were tetracycline-susceptible. All 53 fluoroquinolone-resistant isolates contained ParC and/or GyrA substitutions. Resistances to rifampin (eight isolates), trimethoprim, chloramphenicol and vancomycin (two isolates each) were predicted by the pipeline. Resistance to macrolides/lincosamides without pipeline prediction was rare and correlated to divergent resistance genes or rRNA A2062G substitution. A selection of 267 isolates assigned WGS-based serotypes were also conventionally serotyped. Of these, 246 (92.1%) were in agreement, with the remaining 21 (7.8%) conventionally non-serotypeable. For 32 of 1975 isolates (1.6%), WGS-based serotypes could not be assigned. CONCLUSION: The WGS-based assignment of iGBS resistance features and serotypes is an accurate substitute for phenotypic testing.

Using whole genome sequencing to identify resistance determinants and predict antimicrobial resistance phenotypes for year 2015 invasive pneumococcal disease isolates recovered in the United States.

Our whole genome sequence (WGS) pipeline was assessed for accurate prediction of antimicrobial phenotypes. For 2316 invasive pneumococcal isolates recovered during 2015 we compared WGS pipeline data to broth dilution testing (BDT) for 18 antimicrobials. For 11 antimicrobials categorical discrepancies were assigned when WGS-predicted MICs and BDT MICs predicted different categorizations for susceptibility, intermediate resistance or resistance, ranging from 0.9% (tetracycline) to 2.9% (amoxicillin). For ß-lactam antibiotics, the occurrence of at least four-fold differences in MIC ranged from 0.2% (meropenem) to 1.0% (penicillin), although phenotypic retesting resolved 25%-78% of these discrepancies. Non-susceptibility to penicillin, predicted by penicillin-binding protein types, was 2.7% (non-meningitis criteria) and 23.8% (meningitis criteria). Other common resistance determinants included mef (475 isolates), ermB (191 isolates), ermB + mef (48 isolates), tetM (261 isolates) and cat (51 isolates). Additional accessory resistance genes (tetS, tet32, aphA-3, sat4) were rarely detected (one to three isolates). Rare core genome mutations conferring erythromycin-resistance included a two-codon rplD insertion (rplD69-KG-70) and the 23S rRNA A2061G substitution (six isolates). Intermediate cotrimoxazole-resistance was associated with one or two codon insertions within folP (238 isolates) or the folA I100L substitution (38 isolates), whereas full cotrimoxazole-resistance was attributed to alterations in both genes (172 isolates). The two levofloxacin-resistant isolates contained parC and/or gyrA mutations. Of 11 remaining isolates with moderately elevated MICs to both ciprofloxacin and levofloxacin, seven contained parC or gyrA mutations. The two rifampin-resistant isolates contained rpoB mutations. WGS-based antimicrobial phenotype prediction was an informative alternative to BDT for invasive pneumococci.

Strain features and distributions in pneumococci from children with invasive disease before and after 13-valent conjugate vaccine implementation in the USA.

The effect of second-generation pneumococcal conjugate vaccines on invasive pneumococcal disease (IPD) strain distributions have not yet been well described. We analysed IPD isolates recovered from children aged <5 years through Active Bacterial Core surveillance before (2008-2009; n = 828) and after (2011-2013; n = 600) 13-valent pneumococcal conjugate vaccine (PCV13) implementation. We employed conventional testing, PCR/electrospray ionization mass spectrometry and whole genome sequence (WGS) analysis to identify serotypes, resistance features, genotypes, and pilus types. PCV13, licensed in February 2010, effectively targeted all major 19A and 7F genotypes, and decreased antimicrobial resistance, primarily owing to removal of the 19A/ST320 complex. The strain complex contributing most to the remaining ß-lactam resistance during 2011-2013 was 35B/ST558. Significant emergence of non-vaccine clonal complexes was not evident. Because of the removal of vaccine serotype strains, positivity for one or both pilus types (PI-1 and PI-2) decreased in the post-PCV13 years 2011-2013 relative to 2008-2009 (decreases of 32-55% for PI-1, and >95% for PI-2 and combined PI-1 + PI-2). ß-Lactam susceptibility phenotypes correlated consistently with transpeptidase region sequence combinations of the three major penicillin-binding proteins (PBPs) determined through WGS analysis. Other major resistance features were predictable by DNA signatures from WGS analysis. Multilocus sequence data combined with PBP combinations identified progeny, serotype donors and recipient strains in serotype switch events. PCV13 decreased the frequency of all PCV13 serotype clones and concurrently decreased the frequency of strain subsets with resistance and/or adherence features conducive to successful carriage. Our results serve as a reference describing key features of current paediatric IPD strains in the USA after PCV13 implementation.

Tobacco use among adults with disabilities in Massachusetts.

OBJECTIVES: To examine the characteristics of smoking among adults with disabilities in Massachusetts. DESIGN: Data were obtained from the 1996-1999 Massachusetts Behavioral Risk Factor Surveillance System, a random digit dial telephone survey. Respondents reporting use of special equipment or a limitation caused by impairment or health problem were classified as having a disability. Adults with disabilities were further classified by level, based on need for assistance, and type of disability. Logistic regression models were used to assess the association between disability status and smoking. SETTING AND PARTICIPANTS: Random sample of non-institutionalised Massachusetts adults, 18 and older, with disabilities (n = 2985) and without disabilities (n = 14 395). MAIN OUTCOME MEASURES: Smoking status, intensity, and factors related to quitting. RESULTS: Compared to those without disabilities, adults with disabilities were more likely to have ever smoked (odds ratio (OR) 1.42, 95% confidence interval (CI) 1.25 to 1.61) and to be current smokers (OR 1.52, 95% CI 1.32 to 1.76). Smoking rates varied by type of disability. Among current smokers, adults with disabilities smoked more cigarettes per day (OR 1.65, 95% CI 1.31 to 2.16), sooner after waking (OR 1.50, 95% CI 1.13 to 1.99), and were more likely to be advised by a doctor to quit (OR 2.07, 95% CI 1.60 to 2.69). Adults with disabilities who needed assistance were more likely to be planning to quit (OR 1.50, 95% CI 0.99 to 2.26). CONCLUSIONS: There are disparities in smoking rates between adults with and without disabilities. Smoking cessation programmes targeted to the disabled community are needed.

Macrophage colony-stimulating factor mediates invasion of ovarian cancer cells through urokinase.

The macrophage colony-stimulating factor (CSF-1) is best known as a hematopoietic cytokine important to macrophage activation. Recently, the importance of CSF-1 and its receptor (encoded by the c-fms proto-oncogene) in epithelial ovarian cancer has also been recognized, with overexpression of CSF-1 denoting poor prognosis in ovarian cancer patients. During macrophage activation, CSF-1 promotes urokinase-type plasminogen activator (uPA) activity; in macrophages and in malignant cells of lung, breast, colon, and prostatic origin, uPA activity is strongly correlated with the ability to invade and, in the malignant cells, to metastasize. While there is clear evidence of CSF-1 and uPA expression in primary and metastatic ovarian cancer, the significance of their expression to invasion of these cells has not been explored. We find that all of our ovarian cancer cell lines which we have studied co-express CSF-1 and uPA transcripts and protein. Urokinase expression in these ovarian cancer cell lines correlates with the degree of tumorigenicity in nude mice, with the most virulent tumor resulting from Hey cells, a strong expressor of uPA. We studied the invasion of these primary and established ovarian cancer cells through a Matrigel (reconstituted basement membrane matrix) barrier. The ability of ovarian cancer cells to invade is strongly correlated with endogenous CSF-1 expression (Pearson's correlation, r = 0.91; P = 0.01). A total of 0.90 +/- 0.16% of Bix3 cells (very weak expressor of CSF-1) invaded through the barrier, in contrast to 6.95 +/- 0.75% of Hey cells (strong CSF-1 expressor) and 10.44 +/- 2.33% of Bixler cells (the strongest CSF-1 expressor). We studied the ability of two of the cell lines to invade human laminin and type IV collagen (Bix3, a weak invader of Matrigel, and Hey, a strong invader), to determine (a) whether our results on a Matrigel matrix may represent a relevant model for invasion in humans and (b) whether there is a potential confounding effect from the cytokines and proteases in Matrigel. On this human simple matrix, we confirm that Bix3 is a weakly invasive cell line (0.33 +/- 0.04% invasion) which contrasted to the strongly invasive Hey cell line (8.51 +/- 0.47%). Treatment of Bix3 cells with exogenous CSF-1 stimulates percentage of invasion by 2-fold and results in a similar increase in the level of uPA transcripts and cellular associated uPA antigen. Furthermore, cell surface-bound uPA increased from 74% in the absence of CSF-1 to 100% (fully saturated) in the presence of CSF-1.(ABSTRACT TRUNCATED AT 400 WORDS)

The significance of urokinase- type plasminogen activator, its inhibitors, and its receptor in ascites of patients with epithelial ovarian cancer.

BACKGROUND: Strong correlations have been reported between expression of urokinase-type plasminogen activator (uPA) and poor prognosis in several human carcinomas. The clinical significance of secreted levels of uPA, its receptor (uPAR), and its inhibitors (PAI-1 and PAI-2) in the ascites of patients with epithelial ovarian cancer patients was investigated. METHODS: uPA, uPAR, PAI-1, and PAI-2 were measured in the primary ascites of 36 patients with epithelial ovarian cancer by enzyme-linked immunosorbent assay, and normalized to protein content. The relationship between levels of these factors in ascites and clinicopathologic variables, survival, and disease free interval (DFI) was studied. Because high levels of macrophage colony stimulating factor (CSF-1) in ascites is a known poor prognostic indicator for ovarian cancer, the effect of CSF-1 on secretion of PAI-2 by ovarian cancer cells was also examined in vitro. RESULTS: High uPA levels per se did not correlate with either survival or DFI. However, the ratio of uPAR normalized for uPA content correlated inversely with FIGO stage and residual disease, with a significant association between high uPAR/uPA level and prolonged survival and DFI. High PAI-1 levels (> 0.120 ng/mg) proved to be a good prognostic factor, correlating with both prolonged DFI and residual disease < or = 5 cm among Stage 3 patients. Conversely, high levels of PAI-2 (> 0.061 ng/mg) indicated a poor prognosis in Stage 3 patients, as high PAI-2 levels were associated with shorter DFI and survival. CSF-1 stimulated the secretion of PAI-2 by 2.4-fold in Bix3 ovarian carcinoma cells. Multivariate analysis revealed that the two independent prognostic factors for DFI in Stage 3 patients were PAI-1 and PAI-2; Stage 3 patients with high secreted PAI-1 and low PAI-2 levels have a median DFI that was prolonged by 30 months relative to the rest of the group. CONCLUSION: Secreted uPAR/uPA appears to measure tumor burden and predicts prolonged DFI and survival, but was not found to be an independent prognostic factor on multivariate analysis. High levels of PAI-1 in ascites were independently associated with prolonged DFI among Stage 3 patients. Therefore, secreted uPAR and PAI-1 may modulate uPA activity and lead to improved outcome. This contrasts with the finding that secreted PAI-2 is an independent factor indicating poor prognosis, which may relate in part to the actions of CSF-1. This study emphasizes the importance of uPA, its receptor, and its inhibitors in patients with epithelial ovarian cancer.