A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq

© 2016 Burke & Darling. Background: The bacterial 16S rRNA gene has historically been used in defining bacterial taxonomy and phylogeny. However, there are currently no high-throughput methods to sequence full-length 16S rRNA genes present in a sample with precision. Results: We describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Proof of principle of the approach is demonstrated, with the generation of 1,604 sequences greater than 1,300 nt from a single Nano MiSeq run, with accuracy estimated to be 100-fold higher than standard Illumina reads. The reads were chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection. Conclusions: This method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms. This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution

Before the whistle blows: developing new paradigms in tuberculosis screening to maximise benefit and minimise harm [version 1; peer review: awaiting peer review]

We summarise recent emerging evidence around tuberculosis (TB) transmission and its role in tuberculosis epidemiology, and in novel TB screening and diagnostic tests that will likely become available in low-resource settings in the near future. Little consideration has been paid to how these novel new tests will be implemented, nor what the consequences for individuals, communities and health systems will be. In particular, because of low specificity and consequent false-positive diagnoses, and the low percentage of people who “screen positive” that will go onto develop active pulmonary disease, there is significant potential for inappropriate initiation of TB treatment, as well as stigmatisation, loss of livelihoods and in some setting institutionalisation, with uncertain benefit for individual health or community transmission. We use analogy to prompt consideration of how and where new TB screening tests could be implemented in TB screening programmes in low-resource settings. Acceptance and confidence in TB screening programmes depends on well-functioning public health programmes that use screening algorithms that minimise harms and balance population benefits with autonomy and respect for individuals. Before new TB screening tests and algorithms are introduced, more evidence for their effectiveness, costs, benefits and harms under real-world conditions are required

Near full-length 16S rRNA gene next-generation sequencing revealed Asaia as a common midgut bacterium of wild and domesticated Queensland fruit fly larvae

BACKGROUND: Gut microbiota affects tephritid (Diptera: Tephritidae) fruit fly development, physiology, behavior, and thus the quality of flies mass-reared for the sterile insect technique (SIT), a target-specific, sustainable, environmentally benign form of pest management. The Queensland fruit fly, Bactrocera tryoni (Tephritidae), is a significant horticultural pest in Australia and can be managed with SIT. Little is known about the impacts that laboratory-adaptation (domestication) and mass-rearing have on the tephritid larval gut microbiome. Read lengths of previous fruit fly next-generation sequencing (NGS) studies have limited the resolution of microbiome studies, and the diversity within populations is often overlooked. In this study, we used a new near full-length (> 1300 nt) 16S rRNA gene amplicon NGS approach to characterize gut bacterial communities of individual B. tryoni larvae from two field populations (developing in peaches) and three domesticated populations (mass- or laboratory-reared on artificial diets). RESULTS: Near full-length 16S rRNA gene sequences were obtained for 56 B. tryoni larvae. OTU clustering at 99% similarity revealed that gut bacterial diversity was low and significantly lower in domesticated larvae. Bacteria commonly associated with fruit (Acetobacteraceae, Enterobacteriaceae, and Leuconostocaceae) were detected in wild larvae, but were largely absent from domesticated larvae. However, Asaia, an acetic acid bacterium not frequently detected within adult tephritid species, was detected in larvae of both wild and domesticated populations (55 out of 56 larval gut samples). Larvae from the same single peach shared a similar gut bacterial profile, whereas larvae from different peaches collected from the same tree had different gut bacterial profiles. Clustering of the Asaia near full-length sequences at 100% similarity showed that the wild flies from different locations had different Asaia strains. CONCLUSIONS: Variation in the gut bacterial communities of B. tryoni larvae depends on diet, domestication, and horizontal acquisition. Bacterial variation in wild larvae suggests that more than one bacterial species can perform the same functional role; however, Asaia could be an important gut bacterium in larvae and warrants further study. A greater understanding of the functions of the bacteria detected in larvae could lead to increased fly quality and performance as part of the SIT

Activation of Ventral Tegmental Area 5-HT2C Receptors Reduces Incentive Motivation

FUNDING AND DISCLOSURE The research was funded by Wellcome Trust (WT098012) to LKH; and National Institute of Health (DK056731) and the Marilyn H. Vincent Foundation to MGM. The University of Michigan Transgenic Core facility is partially supported by the NIH-funded University of Michigan Center for Gastrointestinal Research (DK034933). The remaining authors declare no conflict of interest. ACKNOWLEDGMENTS We thank Dr Celine Cansell, Ms Raffaella Chianese and the staff of the Medical Research Facility for technical assistance. We thank Dr Vladimir Orduña for the scientific advice and technical assistance.Peer reviewedPublisher PD

Human African trypanosomiasis amongst urban residents in Kinshasa: a case-control study.

BACKGROUND: Increasing numbers of human African trypanosomiasis (HAT) cases have been reported in urban residents of Kinshasa, Democratic Republic Congo since 1996. We set up a case-control study to identify risk factors for the disease. METHODS: All residents of the urban part of Kinshasa with parasitologically confirmed HAT and presenting for treatment to the city's specialized HAT clinics between 1 August, 2002 and 28 February, 2003 were included as cases. We defined the urban part as the area with contiguous habitation and a population density >5000 inhabitants per square kilometre. A digital map of the area was drawn based on a satellite image. For each case, two serologically negative controls were selected, matched on age, sex and neighbourhood. Logistic regression models were fitted to control for confounding. RESULTS: The following risk factors were independently associated with HAT: travel, commerce and cultivating fields in Bandundu, and commerce and cultivating fields in the rural part of Kinshasa. No association with activities in the city itself was found. DISCUSSION: In 2002, the emergence of HAT in urban residents of Kinshasa appears mainly linked to disease transmission in Bandundu and rural Kinshasa. We recommend to intensify control of these foci, to target HAT screening in urban residents to people with contact with these foci, to increase awareness of HAT amongst health workers in the urban health structures and to strengthen disease surveillance

Honey can inhibit and eliminate biofilms produced by Pseudomonas aeruginosa

© 2019, The Author(s). Chronic wound treatment is becoming increasingly difficult and costly, further exacerbated when wounds become infected. Bacterial biofilms cause most chronic wound infections and are notoriously resistant to antibiotic treatments. The need for new approaches to combat polymicrobial biofilms in chronic wounds combined with the growing antimicrobial resistance crisis means that honey is being revisited as a treatment option due to its broad-spectrum antimicrobial activity and low propensity for bacterial resistance. We assessed four well-characterised New Zealand honeys, quantified for their key antibacterial components, methylglyoxal, hydrogen peroxide and sugar, for their capacity to prevent and eradicate biofilms produced by the common wound pathogen Pseudomonas aeruginosa. We demonstrate that: (1) honey used at substantially lower concentrations compared to those found in honey-based wound dressings inhibited P. aeruginosa biofilm formation and significantly reduced established biofilms; (2) the anti-biofilm effect of honey was largely driven by its sugar component; (3) cells recovered from biofilms treated with sub-inhibitory honey concentrations had slightly increased tolerance to honey; and (4) honey used at clinically obtainable concentrations completely eradicated established P. aeruginosa biofilms. These results, together with their broad antimicrobial spectrum, demonstrate that manuka honey-based wound dressings are a promising treatment for infected chronic wounds, including those with P. aeruginosa biofilms

Antibiotic-specific differences in the response of Staphylococcus aureus to treatment with antimicrobiala combined with manuka honey

Skin infections caused by antibiotic resistant Staphylococcus aureus are a significant health problem worldwide; often associated with high treatment cost and mortality rate. Complex natural products like New Zealand (NZ) manuka honey have been revisited and studied extensively as an alternative to antibiotics due to their potent broad-spectrum antimicrobial activity, and the inability to isolate honey-resistant S. aureus. Previous studies showing synergistic effects between manuka-type honeys and antibiotics have been demonstrated against the growth of one methicillin-resistant S. aureus (MRSA) strain. We have previously demonstrated strong synergistic activity between NZ manuka-type honey and rifampicin against growth and biofilm formation of multiple S. arueus strains. Here, we have expanded our investigation using multiple S. aureus strains and four different antibiotics commonly used to treat S. aureus-related skin infections: rifampicin, oxacillin, gentamicin, and clindamycin. Using checkerboard microdilution and agar diffusion assays with S. aureus strains including clinical isolates and MRSA we demonstrate that manuka-type honey combined with these four antibiotics frequently produces a synergistic effect. In some cases when synergism was not observed, there was a significant enhancement in antibiotic susceptibility. Some strains that were highly resistant to an antibiotic when present alone become sensitive to clinically achievable concentrations when combined with honey. However, not all of the S. aureus strains tested responded in the same way to these combinational treatments. Our findings support the use of NZ manuka-type honeys in clinical treatment against S. aureus-related infections and extend their potential use as an antibiotic adjuvant in combinational therapy. Our data also suggest that manuka-type honeys may not work as antibiotic adjuvants for all strains of S. aureus, and this may help determine the mechanistic processes behind honey synergy

A longitudinal study of the diabetic skin and wound microbiome

© 2017 Gardiner et al. Type II diabetes is a chronic health condition which is associated with skin conditions including chronic foot ulcers and an increased incidence of skin infections. The skin microbiome is thought to play important roles in skin defence and immune functioning. Diabetes affects the skin environment, and this may perturb skin microbiome with possible implications for skin infections and wound healing. This study examines the skin and wound microbiome in type II diabetes. Methods. Eight type II diabetic subjects with chronic foot ulcers were followed over a time course of 10 weeks, sampling from both foot skin (swabs) and wounds (swabs and debrided tissue) every two weeks. A control group of eight control subjects was also followed over 10 weeks, and skin swabs collected from the foot skin every two weeks. Samples were processed for DNA and subject to 16S rRNA gene PCR and sequencing of the V4 region. Results. The diabetic skin microbiome was significantly less diverse than control skin. Community composition was also significantly different between diabetic and control skin, however the most abundant taxa were similar between groups, with differences driven by very low abundant members of the skin communities. Chronic wounds tended to be dominated by the most abundant skin Staphylococcus, while other abundant wound taxa differed by patient. No significant correlations were found between wound duration or healing status and the abundance of any particular taxa. Discussion. The major difference observed in this study of the skin microbiome associated with diabetes was a significant reduction in diversity. The long-term effects of reduced diversity are not yet well understood, but are often associated with disease conditions