Tracking down human contamination in ancient human teeth

DNA contamination arising from the manipulation of ancient calcified tissue samples is a poorly understood, yet fundamental, problem that affects the reliability of ancient DNA (aDNA) studies. We have typed the mitochondrial DNA hypervariable region I of the only 6 people involved in the excavation, washing, and subsequent anthropological and genetic study of 23 Neolithic remains excavated from Granollers (Barcelona, Spain) and searched for their presence among the 572 clones generated during the aDNA analyses of teeth from these samples. Of the cloned sequences, 17.13% could be unambiguously identified as contaminants, with those derived from the people involved in the retrieval and washing of the remains present in higher frequencies than those of the anthropologist and genetic researchers. This finding confirms, for the first time, previous hypotheses that teeth samples are most susceptible to contamination at their initial excavation. More worrying, the cloned contaminant sequences exhibit substitutions that can be attributed to DNA damage after the contamination event, and we demonstrate that the level of such damage increases with time: contaminants that are >10 years old have approximately 5 times more damage than those that are recent. Furthermore, we demonstrate that in this data set, the damage rate of the old contaminant sequences is indistinguishable from that of the endogenous DNA sequences. As such, the commonly used argument that miscoding lesions observed among cloned aDNA sequences can be used to support data authenticity is misleading in scenarios where the presence of old contaminant sequences is possible. We argue therefore that the typing of those involved in the manipulation of the ancient human specimens is critical in order to ensure that generated results are accurate.     

Tracking down the elusive early endosome

Despite significant progress in understanding protein trafficking and compartmentation in plants, the identification and protein compartmentalization for organelles that belong to both the secretory and endocytic pathways have been difficult because protein trafficking has generally been studied separately in these two pathways. However, recent data indicate that the trans-Golgi network serves as an early endosome merging the secretory and endocytic pathways in plant cells. Here, we discuss the proteins identified as markers for post-Golgi compartments in these two pathways and propose that the trans-Golgi network is a pivotal organelle with multiple sorting domains for post-Golgi protein trafficking in plant cells.     

The effect of anthropogenic arsenic contamination on the earthworm microbiome

Earthworms are globally distributed and perform essential roles for soil health and microbial structure. We have investigated the effect of an anthropogenic contamination gradient on the bacterial community of the keystone ecological species Lumbricus rubellus through utilizing 16S rRNA pyrosequencing for the first time to establish the microbiome of the host and surrounding soil. The earthworm‐associated microbiome differs from the surrounding environment which appears to be a result of both filtering and stimulation likely linked to the altered environment associated with the gut micro‐habitat (neutral pH, anoxia and increased carbon substrates). We identified a core earthworm community comprising Proteobacteria (～50%) and Actinobacteria (～30%), with lower abundances of Bacteroidetes (～6%) and Acidobacteria (～3%). In addition to the known earthworm symbiont (Verminephrobacter sp.), we identified a potential host‐associated Gammaproteobacteria species (Serratia sp.) that was absent from soil yet observed in most earthworms. Although a distinct bacterial community defines these earthworms, clear family‐ and species‐level modification were observed along an arsenic and iron contamination gradient. Several taxa observed in uncontaminated control microbiomes are suppressed by metal/metalloid field exposure, including eradication of the hereto ubiquitously associated Verminephrobacter symbiont, which raises implications to its functional role in the earthworm microbiome.     

Tracking human sewage microbiome in a municipal wastewater treatment plant

Human sewage pollution is a major threat to public health because sewage always comes with pathogens. Human sewage is usually received and treated by wastewater treatment plants (WWTPs) to control pathogenic risks and ameliorate environmental health. However, untreated sewage that flows into water environments may cause serious waterborne diseases, as reported in India and Bangladesh. To examine the fate of the human sewage microbiome in a local municipal WWTP of Hong Kong, we used massively parallel sequencing of 16S rRNA gene to systematically profile microbial communities in samples from three sections (i.e., influent, activated sludge, and effluent) obtained monthly throughout 1 year. The results indicated that: (1) influent sewage bacterial profile reflected the human microbiome; (2) human gut bacterial community was the dominant force shaping influent sewage bacterial profile; (3) most human sewage bacteria could be effectively removed by the WWTP; (4) a total of 75 genera were profiled as potentially pathogenic bacteria, most of which were still present in the effluent although at a very low level; (5) a grouped pattern of bacterial community was observed among the same section samples but a dispersed pattern was found among the different section samples; and (6) activated sludge was less affected by the influent sewage bacteria, but it showed a significant impact on the effluent bacteria. All of these findings provide novel insights toward a mechanistic understanding of the fate of human sewage microbiome in the WWTP.     

Distribution and sources of microbial contamination on beef carcasses

Three beef dressing lines of different capacity (160, 440 and 800 head d(-1)) were investigated with respect to contamination associated with carcass/hide and carcass/faeces contacts, the distribution of microbial contamination on carcasses and the antimicrobial efficacy of cold water carcass washes. Swab samples were taken from up to 17 sites for determination of Aerobic Plate Counts at 37 degrees C (APC 37 degrees C) and Escherichia coli enumeration using the Petrifilm procedure. The three beef dressing systems produced virtually identical patterns of microbial contamination. High contamination was found at those sites associated with opening cuts and/or subject to hide contact during hide removal. Where contamination is intermittent, the use of mean microbial data tended to obscure evidence of faecal or hide contact. Consequently, worst-case results, as represented by the 95th percentile value, were used to identify probable instances and sources of contact contamination. Sites not subject to faecal contamination or hide contact typically had swab sample APC (37 degrees C) values of less than log 2.00 cfu cm(-2) accompanied by the occasional detection of E. coli at levels below log 1.00 cfu cm(-2). Sites contacted by 'clean' hide typically had APC (37 degrees C) counts of log 3.00 cfu cm(-2) or greater accompanied by occasional E. coli counts not exceeding log 2.00 cfu cm(-2). Sites contaminated by direct faecal contact or contact with faecally contaminated hides typically had APC (37 degrees C) counts equal to, or greater than, log 4.00 cfu cm(-2) accompanied by E. coli counts exceeding log 2.00 cfu cm(-2). Cold water carcass washing was ineffective in removing microbial contamination and tended to bring about a posterior to anterior redistribution, resulting in increased counts at forequarter sites.     

Top‐down systems biology integration of conditional prebiotic modulated transgenomic interactions in a humanized microbiome mouse model

Gut microbiome–host metabolic interactions affect human health and can be modified by probiotic and prebiotic supplementation. Here, we have assessed the effects of consumption of a combination of probiotics (Lactobacillus paracasei or L. rhamnosus) and two galactosyl‐oligosaccharide prebiotics on the symbiotic microbiome–mammalian supersystem using integrative metabolic profiling and modeling of multiple compartments in germ‐free mice inoculated with a model of human baby microbiota. We have shown specific impacts of two prebiotics on the microbial populations of HBM mice when co‐administered with two probiotics. We observed an increase in the populations of Bifidobacterium longum and B. breve, and a reduction in Clostridium perfringens, which were more marked when combining prebiotics with L. rhamnosus. In turn, these microbial effects were associated with modulation of a range of host metabolic pathways observed via changes in lipid profiles, gluconeogenesis, and amino‐acid and methylamine metabolism associated to fermentation of carbohydrates by different bacterial strains. These results provide evidence for the potential use of prebiotics for beneficially modifying the gut microbial balance as well as host energy and lipid homeostasis.     

Gut microbiome composition and function in experimental colitis during active disease and treatment-induced remission

Dysregulated immune responses to gut microbes are central to inflammatory bowel disease (IBD), and gut microbial activity can fuel chronic inflammation. Examining how IBD-directed therapies influence gut microbiomes may identify microbial community features integral to mitigating disease and maintaining health. However, IBD patients often receive multiple treatments during disease flares, confounding such analyses. Preclinical models of IBD with well-defined disease courses and opportunities for controlled treatment exposures provide a valuable solution. Here, we surveyed the gut microbiome of the T-bet^−/− Rag2^−/− mouse model of colitis during active disease and treatment-induced remission. Microbial features modified among these conditions included altered potential for carbohydrate and energy metabolism and bacterial pathogenesis, specifically cell motility and signal transduction pathways. We also observed an increased capacity for xenobiotics metabolism, including benzoate degradation, a pathway linking host adrenergic stress with enhanced bacterial virulence, and found decreased levels of fecal dopamine in active colitis. When transferred to gnotobiotic mice, gut microbiomes from mice with active disease versus treatment-induced remission elicited varying degrees of colitis. Thus, our study provides insight into specific microbial clades and pathways associated with health, active disease and treatment interventions in a mouse model of colitis.     

Multiple sources of contamination in samples from patients reported to have XMRV infection

Xenotropic murine leukemia virus (MLV)-related retrovirus (XMRV) was reported to be associated with prostate cancer by Urisman, et al. in 2006 and chronic fatigue syndrome (CFS) by Lombardi, et al. in 2009. To investigate this association, we independently evaluated plasma samples from 4 patients with CFS reported by Lombardi, et al. to have XMRV infection and from 5 healthy controls reported to be XMRV uninfected. We also analyzed viral sequences obtained from supernatants of cell cultures found to contain XMRV after coculture with 9 clinical samples from 8 patients. A qPCR assay capable of distinguishing XMRV from endogenous MLVs showed that the viral sequences detected in the CFS patient plasma behaved like endogenous MLVs and not XMRV. Single-genome sequences (N = 89) from CFS patient plasma were indistinguishable from endogenous MLVs found in the mouse genome that are distinct from XMRV. By contrast, XMRV sequences were detected by qPCR in 2 of the 5 plasma samples from healthy controls (sequencing of the qPCR product confirmed XMRV not MLV). Single-genome sequences (N = 234) from the 9 culture supernatants reportedly positive for XMRV were indistinguishable from XMRV sequences obtained from 22Rv1 and XMRV-contaminated 293T cell-lines. These results indicate that MLV DNA detected in the plasma samples from CFS patients evaluated in this study was from contaminating mouse genomic DNA and that XMRV detected in plasma samples from healthy controls and in cultures of patient samples was due to cross-contamination with XMRV (virus or nucleic acid).     

Inactivation of indicator micro-organisms from various sources of faecal contamination in seawater and freshwater

AIM: The survival of indicator micro-organisms in aquatic systems is affected by both biotic and abiotic factors. Much of the past research on this topic has been conducted using laboratory-generated cultures of indicator bacteria. For this study, we used natural sources of faecal contamination as inoculants into environmental water samples, thereby representing the wide diversity of organisms likely to be found in faecal contamination. METHODS AND RESULTS: Rates of inactivation of water quality indicators, total coliforms (TC), Escherichia coli, enterococci (EC) and F+-specific coliphage were studied in three experiments using inoculants of sewage influent, sewage effluent and urban storm drain run-off. Effects of temperature, nutrients, total suspended solids, bacterial load and solar irradiation were studied in fresh and seawater matrices. Results demonstrated that temperature and solar irradiation had significant effects upon rates of inactivation (anova, P < 0.001). Inactivation rates were similar, regardless of the inoculant type. EC degraded the slowest in the dark with T90s of 115-121 and 144-177 h at 20 and 14 degrees C, respectively. When incubated in sunlight, EC was inactivated significantly more rapidly than either E. coli or F+-specific coliphage (P < 0.001). CONCLUSIONS: Inactivation of indicator bacteria is not dependent upon the original source of contamination. Inactivation rates of indicator bacteria were similar in fresh and seawater matrices. However, EC degraded more rapidly in sunlight than E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the source of faecal contamination is not an important factor to inactivation rates of indicator bacteria. However, rates of inactivation of indicator bacteria are likely system specific.     

Antibiotic resistance indexing of Escherichia coli to identify sources of fecal contamination in water

A total of 202 Escherichia coli isolated from urban and rural water were tested with 11 antibiotics to assess the prevalence of antibiotic resistance from each source. Urban waters harbored higher percentages of resistant E. coli strains than rural waters. Antibiotic-resistant E. coli may offer an index of water quality related to source.     

Rapid identification and differentiation of agricultural faecal contamination sources using multiplex PCR

Aims:  To develop a quick, easy-to-use, robust and sensitive multiplex PCR assay to detect common sources of agricultural faecal contamination using a combination of bacterial and eukaryote-specific PCR targets.
Method and Results:  A novel multiplex PCR method was developed that utilizes primers specific for a conserved region of the eukaryote cytochrome-B gene as well as a universal 16S rRNA and the E. coli -specific uidA gene. This multiplex PCR assay was capable of identifying faecal amendments from pig, sheep, cow and goat sources in 24/30 (80%) of amended water samples.
Conclusions:  The method was capable of accurately identifying common agricultural sources.
Significance and Impact of the study:  The procedure described here is simple, rapid (<5 h) and can be used as a first step in microbial source tracking studies, particularly where agricultural faecal contamination is suspected.