Gemma L. Kay

Recovery of a Medieval Brucella melitensis Genome Using Shotgun Metagenomics

ABSTRACT Shotgun metagenomics provides a powerful assumption-free approach to the recovery of pathogen genomes from contemporary and historical material. We sequenced the metagenome of a calcified nodule from the skeleton of a 14th-century middle-aged male excavated from the medieval Sardinian settlement of Geridu. We obtained 6.5-fold coverage of a Brucella melitensis genome. Sequence reads from this genome showed signatures typical of ancient or aged DNA. Despite the relatively low coverage, we were able to use information from single-nucleotide polymorphisms to place the medieval pathogen genome within a clade of B. melitensis strains that included the well-studied Ether strain and two other recent Italian isolates. We confirmed this placement using information from deletions and IS711 insertions. We conclude that metagenomics stands ready to document past and present infections, shedding light on the emergence, evolution, and spread of microbial pathogens. IMPORTANCE Infectious diseases have shaped human populations and societies throughout history. The recovery of pathogen DNA sequences from human remains provides an opportunity to identify and characterize the causes of individual and epidemic infections. By sequencing DNA extracted from medieval human remains through shotgun metagenomics, without target-specific capture or amplification, we have obtained a draft genome sequence of an ~700-year-old Brucella melitensis strain. Using a variety of bioinformatic approaches, we have shown that this historical strain is most closely related to recent strains isolated from Italy, confirming the continuity of this zoonotic infection, and even a specific lineage, in the Mediterranean region over the centuries. Infectious diseases have shaped human populations and societies throughout history. The recovery of pathogen DNA sequences from human remains provides an opportunity to identify and characterize the causes of individual and epidemic infections. By sequencing DNA extracted from medieval human remains through shotgun metagenomics, without target-specific capture or amplification, we have obtained a draft genome sequence of an ~700-year-old Brucella melitensis strain. Using a variety of bioinformatic approaches, we have shown that this historical strain is most closely related to recent strains isolated from Italy, confirming the continuity of this zoonotic infection, and even a specific lineage, in the Mediterranean region over the centuries.

The gut-adherent microbiota of PSC-IBD is distinct to that of IBD.

Primary sclerosing cholangitis–IBD (PSC–IBD) is an inflammatory autoimmune hepato–biliary–enteric disease in which it is predicted that gut microbiota have potential pathophysiological effects, relevant to disease initiation and outcome. The recent article by Kummen et al 1 who reported that the gut microbiota in PSC is distinct compared with those from healthy controls and patients with UC without liver disease, is therefore of interest. However, it remains unclear if these alterations in the gut microbiota are a cause or an effect of liver disease, and there remains a challenging task to link dysbiosis with disease pathogenesis, as well as clarify whether faecal microbiota are entirely representative of communities of mucosa-associated bacteria, which might uniquely interact with immune and epithelial cells. Nevertheless Kummen et al notably demonstrated that the Veillonella genus showed a marked increase in PSC–IBD, in comparison with both healthy controls and patients with UC alone. Given interest in the mechanism of lymphocyte tracking between the bowel and liver …

Differences in the Faecal Microbiome in Schistosoma haematobium Infected Children vs. Uninfected Children

Background Several infectious diseases and therapeutic interventions cause gut microbe dysbiosis and associated pathology. We characterised the gut microbiome of children exposed to the helminth Schistosoma haematobium pre- and post-treatment with the drug praziquantel (PZQ), with the aim to compare the gut microbiome structure (abundance and diversity) in schistosome infected vs. uninfected children. Methods Stool DNA from 139 children aged six months to 13 years old; with S. haematobium infection prevalence of 27.34% was extracted at baseline. 12 weeks following antihelminthic treatment with praziqunatel, stool DNA was collected from 62 of the 139 children. The 16S rRNA genes were sequenced from the baseline and post-treatment samples and the sequence data, clustered into operational taxonomic units (OTUs). The OTU data were analysed using multivariate analyses and paired T- test. Results Pre-treatment, the most abundant phyla were Bacteroidetes, followed by Firmicutes and Proteobacteria respectively. The relative abundance of taxa among bacterial classes showed limited variation by age group or sex and the bacterial communities had similar overall compositions. Although there were no overall differences in the microbiome structure across the whole age range, the abundance of 21 OTUs varied significantly with age (FDR<0.05). Some OTUs including Veillonella, Streptococcus, Bacteroides and Helicobacter were more abundant in children ≤ 1 year old compared to older children. Furthermore, the gut microbiome differed in schistosome infected vs. uninfected children with 27 OTU occurring in infected but not uninfected children, for 5 of these all Prevotella, the difference was statistically significant (p <0.05) with FDR <0.05. PZQ treatment did not alter the microbiome structure in infected or uninfected children from that observed at baseline. Conclusions There are significant differences in the gut microbiome structure of infected vs. uninfected children and the differences were refractory to PZQ treatment.

An In Vitro Chicken Gut Model Demonstrates Transfer of a Multidrug Resistance Plasmid from Salmonella to Commensal Escherichia coli

ABSTRACT The chicken gastrointestinal tract is richly populated by commensal bacteria that fulfill various beneficial roles for the host, including helping to resist colonization by pathogens. It can also facilitate the conjugative transfer of multidrug resistance (MDR) plasmids between commensal and pathogenic bacteria which is a significant public and animal health concern as it may affect our ability to treat bacterial infections. We used an in vitro chemostat system to approximate the chicken cecal microbiota, simulate colonization by an MDR Salmonella pathogen, and examine the dynamics of transfer of its MDR plasmid harboring several genes, including the extended-spectrum beta-lactamase blaCTX-M1. We also evaluated the impact of cefotaxime administration on plasmid transfer and microbial diversity. Bacterial community profiles obtained by culture-independent methods showed that Salmonella inoculation resulted in no significant changes to bacterial community alpha diversity and beta diversity, whereas administration of cefotaxime caused significant alterations to both measures of diversity, which largely recovered. MDR plasmid transfer from Salmonella to commensal Escherichia coli was demonstrated by PCR and whole-genome sequencing of isolates purified from agar plates containing cefotaxime. Transfer occurred to seven E. coli sequence types at high rates, even in the absence of cefotaxime, with resistant strains isolated within 3 days. Our chemostat system provides a good representation of bacterial interactions, including antibiotic resistance transfer in vivo. It can be used as an ethical and relatively inexpensive approach to model dissemination of antibiotic resistance within the gut of any animal or human and refine interventions that mitigate its spread before employing in vivo studies. IMPORTANCE The spread of antimicrobial resistance presents a grave threat to public health and animal health and is affecting our ability to respond to bacterial infections. Transfer of antimicrobial resistance via plasmid exchange is of particular concern as it enables unrelated bacteria to acquire resistance. The gastrointestinal tract is replete with bacteria and provides an environment for plasmid transfer between commensals and pathogens. Here we use the chicken gut microbiota as an exemplar to model the effects of bacterial infection, antibiotic administration, and plasmid transfer. We show that transfer of a multidrug-resistant plasmid from the zoonotic pathogen Salmonella to commensal Escherichia coli occurs at a high rate, even in the absence of antibiotic administration. Our work demonstrates that the in vitro gut model provides a powerful screening tool that can be used to assess and refine interventions that mitigate the spread of antibiotic resistance in the gut before undertaking animal studies. IMPORTANCE The spread of antimicrobial resistance presents a grave threat to public health and animal health and is affecting our ability to respond to bacterial infections. Transfer of antimicrobial resistance via plasmid exchange is of particular concern as it enables unrelated bacteria to acquire resistance. The gastrointestinal tract is replete with bacteria and provides an environment for plasmid transfer between commensals and pathogens. Here we use the chicken gut microbiota as an exemplar to model the effects of bacterial infection, antibiotic administration, and plasmid transfer. We show that transfer of a multidrug-resistant plasmid from the zoonotic pathogen Salmonella to commensal Escherichia coli occurs at a high rate, even in the absence of antibiotic administration. Our work demonstrates that the in vitro gut model provides a powerful screening tool that can be used to assess and refine interventions that mitigate the spread of antibiotic resistance in the gut before undertaking animal studies.

Forensic Analysis Reveals Acute Decompensation of Chronic Heart Failure in a 3500‐Year‐Old Egyptian Dignitary

Naturally preserved and embalmed bodies from archeological contexts represent a powerful source of information for forensic investigators. They allow one to ascertain pathology, cause of death, to enhance diagnostic methodology, and to improve the analysis of altered remains. We investigated the complete head and lung remnants of a 3,500‐year‐old Egyptian dignitary by radiological, microscopic, and genetic approaches. The individual, a middle‐aged male, suffered from severe periodontitis, mild atherosclerosis, and experienced cardiogenic pulmonary insufficiency with recurrent mini‐bleeds and pulmonary edema. Histology and ancient DNA analyses excluded the presence of Mycobacterium tuberculosis or of any other pathogenic species. Based on our collection of evidence, we propose that acute decompensation complicating chronic cardiac insufficiency was the likely cause of death. The underlying causes for this failure remain unknown although chronic hypertension appears to be the most likely candidate. Our finding represents the earliest reported case of chronic heart failure in ancient mummies.

Identification of the anti-mycobacterial functional properties of piperidinol derivatives

Tuberculosis (TB) remains a major global health threat and is now the leading cause of death from a single infectious agent worldwide. The current TB drug regimen is inadequate, and new anti‐tubercular agents are urgently required to be able to successfully combat the increasing prevalence of drug‐resistant TB. The purpose of this study was to investigate a piperidinol compound derivative that is highly active against the Mycobacterium tuberculosis bacillus.

Draft Genome Sequences of Six Novel Bacterial Isolates from Chicken Ceca

ABSTRACT The chicken is the most common domesticated animal and the most abundant bird in the world. However, the chicken gut is home to many previously uncharacterized bacterial taxa. Here, we report draft genome sequences from six bacterial isolates from chicken ceca, all of which fall outside any named species.

Whole-genome sequencing illuminates the evolution and spread of multidrug-resistant tuberculosis in Southwest Nigeria

Nigeria has an emerging problem with multidrug-resistant tuberculosis (MDR-TB). Whole-genome sequencing was used to understand the epidemiology of tuberculosis and genetics of multi-drug resistance among patients from two tertiary referral centers in Southwest Nigeria. In line with previous molecular epidemiology studies, most isolates of Mycobacterium tuberculosis from this dataset belonged to the Cameroon clade within the Euro-American lineage. Phylogenetic analysis showed this clade was undergoing clonal expansion in this region, and suggests that it was involved in community transmission of sensitive and multidrug-resistant tuberculosis. Five patients enrolled for retreatment were infected with pre-extensively drug resistant (pre-XDR) due to fluoroquinolone resistance in isolates from the Cameroon clade. In all five cases resistance was conferred through a mutation in the gyrA gene. In some patients, genomic changes occurred in bacterial isolates during the course of treatment that potentially led to decreased drug susceptibility. We conclude that inter-patient transmission of resistant isolates, principally from the Cameroon clade, contributes to the spread of MDR-TB in this setting, underscoring the urgent need to curb the spread of multi-drug resistance in this region.

PWE-061 Dysbiosis in Patients with Bile Acid Diarrhoea (BAD) Demonstrated Using 16S RNA Gene Sequencing

Introduction The gastrointestinal tract harbours a diverse community of approximately 1014 microorganisms comprising 500 to 1000 bacterial species.1 Dysbiosis occurs when pathological imbalances in the gut bacterial community precipitate disease and has been linked to the dysmetabolism of bile acids (BAs) in the gut. 1–2% of primary BAs escape the enterohepatic circulation and undergo microbial biotransformation in the large bowel to form the secondary BAs, deoxycholic acid (DCA) and lithocholic acid (LCA).2 BA metabolites as a result of microbial transformations act as signalling molecules via the Farnesoid X receptor (FXR). Activation of the ileal FXR stimulates expression of Fibroblast Growth Factor 19 (FGF-19), which binds hepatic FGF-4 and activates JNK1/2 and ERK1/2 to impede the physiological feedback of BA synthesis and maintain a functional BA pool.3 The degree of activation of BA receptors is influenced primarily by the gut microbiota and therefore dysbiosis may result in abnormal BA modification leading to the development of gastrointestinal disease. To enhance our understanding of gut dysbiosis in patients with BAD, 16 S rRNA gene sequencing was undertaken to investigate bacterial communities from faecal samples in patients with BAD and IBS. Methods 35 patients (20 with BAD and 15 with IBS) were recruited from the nuclear medicine department at University Hospitals Coventry and Warwickshire NHS Trust after referral from the gastroenterology clinic for a SeHCAT scan to investigate chronic diarrhoea. The patients with BAD had either Type 1 with a diagnosis of Crohn’s disease, type 2 or type 3 post cholecystectomy. Stool samples were stored at -80oC within two hours of collection and then defrosted for 45 minutes at room temperature prior to analysis. 16 s RNA sequencing was undertaken by isolating DNA from stool samples using the QIAamp Fast DNA Stool Extraction kit. V3-V4 primers and extensor ready mix (Thermo scientific) were used to amplify the 16 s rRNA gene sequences from isolated metagenomic DNA. The eluted DNA was then quantified using the broad-range Qubit kit. DNA was analysed by electrophoresis on 1% agarose gels and purified post-PCR. The DNA was then denatured and sequenced on a MiSeq using the Illumina Miseq V2 2x250 bp paired end protocol. Bioinformatic statistical analysis was undertaken using QUIIME and the UPARSE pipeline. Results The rarefaction plots showed reduced diversity in BAD patients compared with IBS; p = 0.0007216. This was also confirmed using the Shannon’s diversity index. The abundance of certain taxa namely Lachnospiraceae (p = 0.00134), Rumiinococcus (p = 0.0034) and Prevotella (p = 0.0046) were reduced in patients with BAD. Conclusion We have shown that diversity in those with BAD is significantly reduced compared to those with IBS with certain phyla predominating. References 1 Gérard P. Metabolism of Cholesterol and Bile Acids by the Gut Microbiota. Pathogens 2014;3:14–24. 2. Vlahcevic ZR, Heuman DM, Hylemon PB. Physiology and pathophysiology of enterohepatic circulation of bile acids. In D. Zakim and T. Boyer, editors. Hepatology: A Textbook of Liver Disease. 1996, 3rd edition. Vol. 1. Saunders, Philadelphia, PA. 376–417 3 Tsuei J, Chau T, Mills D, et al. Bile acid dysregulation, gut dysbiosis, and gastrointestinal cancer. Exp Biol Med (Maywood). 2014 Nov;239(11):1489–504. Disclosure of Interest None Declared