Tania Contente-Cuomo

Multidrug-Resistant Staphylococcus aureus in US Meat and Poultry

We characterized the prevalence, antibiotic susceptibility profiles, and genotypes of Staphylococcus aureus among US meat and poultry samples (n = 136). S. aureus contaminated 47% of samples, and multidrug resistance was common among isolates (52%). S. aureus genotypes and resistance profiles differed significantly among sample types, suggesting food animal–specific contamination.

Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer

Circulating tumour DNA analysis can be used to track tumour burden and analyse cancer genomes non-invasively but the extent to which it represents metastatic heterogeneity is unknown. Here we follow a patient with metastatic ER-positive and HER2-positive breast cancer receiving two lines of targeted therapy over 3 years. We characterize genomic architecture and infer clonal evolution in eight tumour biopsies and nine plasma samples collected over 1,193 days of clinical follow-up using exome and targeted amplicon sequencing. Mutation levels in the plasma samples reflect the clonal hierarchy inferred from sequencing of tumour biopsies. Serial changes in circulating levels of sub-clonal private mutations correlate with different treatment responses between metastatic sites. This comparison of biopsy and plasma samples in a single patient with metastatic breast cancer shows that circulating tumour DNA can allow real-time sampling of multifocal clonal evolution.

Male Circumcision Significantly Reduces Prevalence and Load of Genital Anaerobic Bacteria

ABSTRACT Male circumcision reduces female-to-male HIV transmission. Hypothesized mechanisms for this protective effect include decreased HIV target cell recruitment and activation due to changes in the penis microbiome. We compared the coronal sulcus microbiota of men from a group of uncircumcised controls (n = 77) and from a circumcised intervention group (n = 79) at enrollment and year 1 follow-up in a randomized circumcision trial in Rakai, Uganda. We characterized microbiota using16S rRNA gene-based quantitative PCR (qPCR) and pyrosequencing, log response ratio (LRR), Bayesian classification, nonmetric multidimensional scaling (nMDS), and permutational multivariate analysis of variance (PerMANOVA). At baseline, men in both study arms had comparable coronal sulcus microbiota; however, by year 1, circumcision decreased the total bacterial load and reduced microbiota biodiversity. Specifically, the prevalence and absolute abundance of 12 anaerobic bacterial taxa decreased significantly in the circumcised men. While aerobic bacterial taxa also increased postcircumcision, these gains were minor. The reduction in anaerobes may partly account for the effects of circumcision on reduced HIV acquisition. IMPORTANCE The bacterial changes identified in this study may play an important role in the HIV risk reduction conferred by male circumcision. Decreasing the load of specific anaerobes could reduce HIV target cell recruitment to the foreskin. Understanding the mechanisms that underlie the benefits of male circumcision could help to identify new intervention strategies for decreasing HIV transmission, applicable to populations with high HIV prevalence where male circumcision is culturally less acceptable. The bacterial changes identified in this study may play an important role in the HIV risk reduction conferred by male circumcision. Decreasing the load of specific anaerobes could reduce HIV target cell recruitment to the foreskin. Understanding the mechanisms that underlie the benefits of male circumcision could help to identify new intervention strategies for decreasing HIV transmission, applicable to populations with high HIV prevalence where male circumcision is culturally less acceptable.

Medical therapy reduces microbiota diversity and evenness in surgically recalcitrant chronic rhinosinusitis

Chronic rhinosinusitis (CRS) is a highly prevalent and heterogeneous condition frequently treated with antibiotics and corticosteroid therapy. However, the effect of medical therapy on sinus microbiota remains unknown.

Rapid differentiation between livestock-associated and livestock-independent Staphylococcus aureus CC398 clades.

Staphylococcus aureus clonal complex 398 (CC398) isolates cluster into two distinct phylogenetic clades based on single-nucleotide polymorphisms (SNPs) revealing a basal human clade and a more derived livestock clade. The scn and tet(M) genes are strongly associated with the human and the livestock clade, respectively, due to loss and acquisition of mobile genetic elements. We present canonical single-nucleotide polymorphism (canSNP) assays that differentiate the two major host-associated S. aureus CC398 clades and a duplex PCR assay for detection of scn and tet(M). The canSNP assays correctly placed 88 S. aureus CC398 isolates from a reference collection into the human and livestock clades and the duplex PCR assay correctly identified scn and tet(M). The assays were successfully applied to a geographically diverse collection of 272 human S. aureus CC398 isolates. The simple assays described here generate signals comparable to a whole-genome phylogeny for major clade assignment and are easily integrated into S. aureus CC398 surveillance programs and epidemiological studies.

The Otologic Microbiome A Study of the Bacterial Microbiota in a Pediatric Patient With Chronic Serous Otitis Media Using 16SrRNA Gene-Based Pyrosequencing

OBJECTIVE To characterize bacterial microbiota in middle ear, adenoid, and tonsil specimens using 16SrRNA gene-based pyrosequencing analysis. DESIGN Cross-sectional study of bacterial microbiota in middle ear, adenoid, and tonsil specimens from a pediatric patient with chronic serous otitis media. Middle ear, adenoid, and tonsil specimens from a pediatric patient were collected and underwent cell lysis and DNA isolation. Pyrosequencing was performed on the 454 Life Sciences GS FLX platform (Roche Diagnostics Corp, Branford, Connecticut). Pyrosequencing data were processed, quality-checked, and taxonomically classified to generate an abundance-based matrix. Ecological analyses were performed. SETTING Academic, tertiary referral center. MAIN OUTCOME MEASURES Comparative microbiome analysis. RESULTS We detected a total of 17 unique bacterial families, with 9, 9, and 12 bacterial families from the middle ear, tonsil, and adenoid specimens, respectively. Pseudomonadaceae dominated the middle ear microbiota at 82.7% relative abundance, whereas Streptococcaceae dominated the tonsil microbiota at 69.2%. Multiple bacteria, including Pseudomonadaceae, Streptococcaceae, Fusobacteriaceae, and Pasteurellaceae, dominated the adenoid microbiota. Overlap between the middle ear and the tonsil microbiota was minimal. In contrast, the adenoid microbiota encompassed bacteria detected from middle ear and tonsil. CONCLUSIONS Bacterial community analysis using pyrosequencing analysis revealed diverse, previously unknown bacterial communities in a set of pediatric middle ear, tonsil, and adenoid specimens. Our findings suggest that the adenoid may be a source site for both the middle ear and tonsil microbiota. An ecological framework is appropriate in comparative analysis of microbiota from nonsterile body sites.

Complete genome sequence of the epidemic and highly virulent CTX-M-15-producing H30-Rx subclone of Escherichia coli ST131

ABSTRACT We report the complete genome sequence, including five complete plasmid sequences, of Escherichia coli ST131 isolate JJ1886. The isolate was obtained in 2007 in the United States from a patient with fatal urosepsis and belongs to the virulent, CTX-M-15-producing H30-Rx sublineage.

The semen microbiome and its relationship with local immunology and viral load in HIV infection.

Semen is a major vector for HIV transmission, but the semen HIV RNA viral load (VL) only correlates moderately with the blood VL. Viral shedding can be enhanced by genital infections and associated inflammation, but it can also occur in the absence of classical pathogens. Thus, we hypothesized that a dysregulated semen microbiome correlates with local HIV shedding. We analyzed semen samples from 49 men who have sex with men (MSM), including 22 HIV-uninfected and 27 HIV-infected men, at baseline and after starting antiretroviral therapy (ART) using 16S rRNA gene-based pyrosequencing and quantitative PCR. We studied the relationship of semen bacteria with HIV infection, semen cytokine levels, and semen VL by linear regression, non-metric multidimensional scaling, and goodness-of-fit test. Streptococcus, Corynebacterium, and Staphylococcus were common semen bacteria, irrespective of HIV status. While Ureaplasma was the more abundant Mollicutes in HIV-uninfected men, Mycoplasma dominated after HIV infection. HIV infection was associated with decreased semen microbiome diversity and richness, which were restored after six months of ART. In HIV-infected men, semen bacterial load correlated with seven pro-inflammatory semen cytokines, including IL-6 (p = 0.024), TNF-α (p = 0.009), and IL-1b (p = 0.002). IL-1b in particular was associated with semen VL (r2 = 0.18, p = 0.02). Semen bacterial load was also directly linked to the semen HIV VL (r2 = 0.15, p = 0.02). HIV infection reshapes the relationship between semen bacteria and pro-inflammatory cytokines, and both are linked to semen VL, which supports a role of the semen microbiome in HIV sexual transmission.

Macroscale spatial variation in chronic wound microbiota : A cross-sectional study

Controlling for sample site is considered to be an important aspect of chronic wound microbiological investigations; yet, macroscale spatial variation in wound microbiota has not been well characterized. A total of 31 curette samples were collected at the leading edge, opposing leading edge, and/or center of 13 chronic wounds. Bacterial community composition was characterized using a combination of 16S rRNA gene‐based pyrosequencing; heat map display; hierarchical clustering; nonmetric multidimensional scaling; and permutation multivariate analysis of variance. A total of 58 bacterial families and 91 bacterial genera were characterized among the 13 wounds. While substantial macroscale spatial variation was observed among the wounds, bacterial communities at different sites within individual wounds were significantly more similar than those in different wounds (p=0.001). Our results support the prevalent opinion that controlling for sample site may improve the quality of wound microbiota studies; however, the significant similarity in bacterial communities from different sites within individual wounds indicates that studies failing to control for sampling site should not be disregarded based solely on this criterion. A composite sample from multiple sites across the surface of individual wounds may provide the most robust characterization of wound microbiota.

Evaluation of pre-analytical factors affecting plasma DNA analysis

Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies.