Dysregulation of macrophage PEPD in obesity determines adipose tissue fibro-inflammation and insulin resistance.

Resulting from impaired collagen turnover, fibrosis is a hallmark of adipose tissue (AT) dysfunction and obesity-associated insulin resistance (IR). Prolidase, also known as peptidase D (PEPD), plays a vital role in collagen turnover by degrading proline-containing dipeptides but its specific functional relevance in AT is unknown. Here we show that in human and mouse obesity, PEPD expression and activity decrease in AT, and PEPD is released into the systemic circulation, which promotes fibrosis and AT IR. Loss of the enzymatic function of PEPD by genetic ablation or pharmacological inhibition causes AT fibrosis in mice. In addition to its intracellular enzymatic role, secreted extracellular PEPD protein enhances macrophage and adipocyte fibro-inflammatory responses via EGFR signalling, thereby promoting AT fibrosis and IR. We further show that decreased prolidase activity is coupled with increased systemic levels of PEPD that act as a pathogenic trigger of AT fibrosis and IR. Thus, PEPD produced by macrophages might serve as a biomarker of AT fibro-inflammation and could represent a therapeutic target for AT fibrosis and obesity-associated IR and type 2 diabetes.

Three phylogenetic groups have driven the recent population expansion of Cryptococcus neoformans.

Cryptococcus neoformans (C. neoformans var. grubii) is an environmentally acquired pathogen causing 181,000 HIV-associated deaths each year. We sequenced 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. The phylogeny of C. neoformans reveals a recent exponential population expansion, consistent with the increase in the number of susceptible hosts. In our study population, this expansion has been driven by three sub-clades of the C. neoformans VNIa lineage; VNIa-4, VNIa-5 and VNIa-93. These three sub-clades account for 91% of clinical isolates sequenced in our study. Combining the genome data with clinical information, we find that the VNIa-93 sub-clade, the most common sub-clade in Uganda and Malawi, was associated with better outcomes than VNIa-4 and VNIa-5, which predominate in Southeast Asia. This study lays the foundation for further work investigating the dominance of VNIa-4, VNIa-5 and VNIa-93 and the association between lineage and clinical phenotype.

Point-prevalence survey of carbapenemase-producing Enterobacteriaceae and vancomycin-resistant enterococci in adult inpatients in a university teaching hospital in the UK.

Infections with carbapenemase-producing Enterobacteriaceae (CPE) and vancomycin-resistant enterococci (VRE) are associated with increased morbidity and mortality, but the carriage rates of CRE and VRE among hospital inpatients are unknown. A point-prevalence survey was conducted to determine CPE and VRE carriage rates in hospitalized adults. Eight hundred and eighteen of 960 (85.2%) adult inpatients were invited to participate in the study. Of these, 595 patients (72.7%) consented and provided specimens. Of 540 samples tested, none were positive for CPE. One hundred and thirty of 540 (24.1%) samples were VRE positive, and 34 of 40 (85%) of wards had cases. Universal screening for CPE may not be cost-effective in low-prevalence settings, but targeted screening of high-risk patients should continue. The optimal screening strategy for VRE remains to be determined, as universal screening and isolation is not feasible in the study setting.

Antimicrobial resistance in human populations: challenges and opportunities.

Antimicrobial resistance (AMR) is a global public health threat. Emergence of AMR occurs naturally, but can also be selected for by antimicrobial exposure in clinical and veterinary medicine. Despite growing worldwide attention to AMR, there are substantial limitations in our understanding of the burden, distribution and determinants of AMR at the population level. We highlight the importance of population-based approaches to assess the association between antimicrobial use and AMR in humans and animals. Such approaches are needed to improve our understanding of the development and spread of AMR in order to inform strategies for the prevention, detection and management of AMR, and to support the sustainable use of antimicrobials in healthcare.

Erratum: Antimicrobial resistance in human populations: challenges and opportunities - ERRATUM.

[This corrects the article DOI: 10.1017/gheg.2017.4.].

Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function.

The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection.

Ability of SPI2 mutant of S. typhi to effectively induce antibody responses to the mucosal antigen enterotoxigenic E. coli heat labile toxin B subunit after oral delivery to humans.

We have evaluated an oral vaccine based on an Salmonella enteric serovar typhi (S. typhi) Ty2 derivative TSB7 harboring deletion mutations in ssaV (SPI-2) and aroC together with a chromosomally integrated copy of eltB encoding the B subunit of enterotoxigenic Escherichia coli heat labile toxin (LT-B) in volunteers. Two oral doses of 10(8) or 10(9)CFU were administered to two groups of volunteers and both doses were well tolerated, with no vaccinemia, and only transient stool shedding. Immune responses to LT-B and S. typhi lipopolysaccharide were demonstrated in 67 and 97% of subjects, respectively, without evidence of anti-carrier immunity preventing boosting of LT-B responses in many cases. Further development of this salmonella-based (spi-VEC) system for oral delivery of heterologous antigens appears warranted.

Subunit vaccines based on intimin and Efa-1 polypeptides induce humoral immunity in cattle but do not protect against intestinal colonisation by enterohaemorrhagic Escherichia coli O157:H7 or O26:H-.

Enterohaemorrhagic Escherichia coli (EHEC) infections in humans are an important public health concern and are commonly acquired via contact with ruminant faeces. Cattle are a key control point however cross-protective vaccines for the control of EHEC in the bovine reservoir do not yet exist. The EHEC serogroups that are predominantly associated with human infection in Europe and North America are O157 and O26. Intimin and EHEC factor for adherence (Efa-1) play important roles in intestinal colonisation of cattle by EHEC and are thus attractive candidates for the development of subunit vaccines. Immunisation of calves with the cell-binding domain of intimin subtypes beta or gamma via the intramuscular route induced antigen-specific serum IgG1 and, in some cases salivary IgA responses, but did not reduce the magnitude or duration of faecal excretion of EHEC O26:H- (Int(280)-beta) or EHEC O157:H7 (Int(280)-gamma) upon subsequent experimental challenge. Similarly, immunisation of calves via the intramuscular route with the truncated Efa-1 protein (Efa-1') from EHEC O157:H7 or a mixture of the amino-terminal and central thirds of the full-length protein (Efa-1-N and M) did not protect against intestinal colonisation by EHEC O157:H7 (Efa-1') or EHEC O26:H- (Efa-1-N and M) despite the induction of humoral immunity. A portion of the serum IgG1 elicited by the truncated recombinant antigens in calves was confirmed to recognise native protein exposed on the bacterial surface. Calves immunised with a mixture of Int(280)-gamma and Efa-1' or an EHEC O157:H7 bacterin via the intramuscular route then boosted via the intranasal route with the same antigens using cholera toxin B subunit as an adjuvant were also not protected against intestinal colonisation by EHEC O157:H7. These studies highlight the need for further studies to develop and test novel vaccines or treatments for control of this important foodborne pathogen.

Universal DNA primers amplify bacterial DNA from human fetal membranes and link Fusobacterium nucleatum with prolonged preterm membrane rupture.

A large number of bacterial species have been identified in fetal membranes after preterm labour (PTL) associated with intrauterine infection by microbiological culture. In this study, we have investigated a molecular and bioinformatic approach to organism identification which surmounts the need for specific and diverse microbiological culture conditions required by conventional methods. Samples of fetal membranes were taken from 37 preterm infants, and 6 normal term controls delivered by caesarean section, in which bacteria had been detected by in situ hybridization of 16S ribosomal RNA using a generic probe. Degenerate primers were designed to amplify bacterial 16S ribosomal DNA by PCR and used to amplify bacterial DNA from human fetal membranes. Amplicons were cloned, sequenced and bacteria were identified bioinformatically by comparison of sequences with known bacterial DNA genomes. In situ hybridization using an organism specific probe was then used to confirm the presence of the commonest identified organism in tissue samples. Bacterial DNA amplified from 15/43 samples, all from preterm deliveries, and the bioinformatic approach identified organisms in all cases. Multiple bacteria were identified including Mycoplasma hominis, Pasturella multocida, Pseudomonas PH1, Escherichia coli and Prevotella bivia. The commonest organism Fusobacterium nucleatum was found in 9/15 (60%) of samples. Ten of the 12 samples obtained after prolonged membrane rupture were positive for bacterial DNA, and 7 of these (70%) contained DNA from F. nucleatum. Bacteria from fetal membranes may be identified by molecular and bioinformatic methods. Further work is warranted to investigate the apparent linkage between F. nucleatum, fetal membrane rupture and preterm delivery.

Shedding patterns of verocytotoxin-producing Escherichia coli strains in a cohort of calves and their dams on a Scottish beef farm.

Rectal fecal samples were taken once a week from 49 calves on the same farm. In addition, the dams of the calves were sampled at the time of calf birth and at the end of the study. Strains of verocytotoxin-producing Escherichia coli (VTEC) were isolated from these samples by using PCR and DNA probe hybridization tests and were characterized with respect to serotype, verocytotoxin gene (vtx) type, and the presence of the intimin (eae) and hemolysin (ehxA) genes. A total of 170 VTEC strains were isolated during 21 weeks from 130 (20%) of 664 samples from calves and from 40 (47%) of 86 samples from their dams. The characteristics of the calf strains differed from those strains isolated from the dams with respect to verocytotoxin 2 and the presence of the eae gene. In addition, no calf shed the same VTEC serogroup (excluding O?) as its dam at birth or at the end of the study. The most frequently detected serogroups in calves were serogroup O26 and provisional serogroup E40874 (VTEC O26 was found in 25 calves), whereas in dams serogroup O91 and provisional serogroup E54071 were the most common serogroups. VTEC O26 shedding appeared to be associated with very young calves and declined as the calves aged, whereas VTEC O2 shedding was associated with housing of the animals. VTEC O26 strains from calves were characterized by the presence of the vtx1, eae, and ehxA genes, whereas vtx2 was associated with VTEC O2 and provisional serogroup E40874. The high prevalence of VTEC O26 and of VTEC strains harboring the eae gene in this calf cohort is notable because of the association of the O26 serogroup and the presence of the eae gene with human disease. No association between calf diarrhea and any of the VTEC serogroups was identified.

Early interactions of Salmonella enterica serovar typhimurium with human small intestinal epithelial explants.

BACKGROUND: Salmonella enterica serovar typhimurium (S typhimurium) causes invasive gastroenteritis in humans, a disease involving significant penetration of the intestinal mucosa. However, few studies have been undertaken to investigate this interaction directly using differentiated human gut tissue. AIMS: To investigate the early interactions of an enteropathogenic strain of S typhimurium with human intestinal mucosa using human intestinal in vitro organ culture (IVOC). METHODS: Wild-type and mutant derivatives of S typhimurium TML were used to compare interactions with cultured human epithelial cells, bovine ligated loops, and human intestinal IVOC. RESULTS: S typhimurium TML was shown to attach to cultured Caco-2 brush border expressing cells and cause tissue damage and fluid accumulation in a ligated bovine loop model.S typhimurium TML bound predominantly to the mucus layer of human IVOC explants during the first four hours of IVOC incubation. From four to eight hours of IVOC incubation, small but characteristic foci of attaching and invading S typhimurium TML were detected as clusters of bacteria interacting with enterocytes, although there was no evidence for large scale invasion of explant tissues. Ruffling of enterocyte membranes associated with adherent Salmonella was visualised using electron microscopy. CONCLUSIONS: Human IVOC can be used as an alternative model for monitoring the interactions between S typhimurium and human intestinal epithelium, thus potentially offering insight into the early stages of human Salmonella induced gastroenteritis.

Subtyping of virulence genes in verocytotoxin-producing Escherichia coli (VTEC) other than serogroup O157 associated with disease in the United Kingdom.

Verocytotoxin-producing Escherichia coli (VTEC) causes a wide spectrum of disease in humans, from mild diarrhoea to haemolytic uraemic syndrome (HUS). The verocytotoxin (vtx) and intimin (eae) genes of VTEC strains, other than those of serogroup O157, were subtyped to identify common properties that may be associated with increased pathogenicity. Strains were isolated from patients with HUS, those with diarrhoea or from asymptomatic individuals. Strains of VTEC that carried vtx(2) gene subtypes vtx(2) and vtx(2c) were most commonly associated with HUS, whereas strains from patients with less severe disease and from the healthy control group were more likely to have vtx(1c) or vtx(2d) genes. The eae gene was detected more frequently in strains isolated from HUS patients than in those associated with cases of diarrhoea; beta-intimin was the most common intimin subtype in strains isolated from both groups of patients. None of the strains from the healthy control group carried the eae gene.

Detection of Escherichia coli serogroups O26, O103, O111 and O145 from bovine faeces using immunomagnetic separation and PCR/DNA probe techniques.

AIMS: The aim of this study was to isolate Escherichia coli O26, O103, O111 and O145 from 745 samples of bovine faeces using (i) immunomagnetic separation (IMS) beads coated with antibodies to lipopolysaccharide, and slide agglutination (SA) tests and (ii) PCR and DNA probes for the detection of the Verocytotoxin (VT) genes. METHODS AND RESULTS: IMS-SA tests detected 132 isolates of presumptive E. coli O26, 112 (85%) were confirmed as serogroup O26 and 102 had the VT genes. One hundred and twenty-two strains of presumptive E. coli O103 were isolated by IMS-SA, 45 (37%) were confirmed as serogroup O103 but only one of these strains was identified as Verocytotoxin-producing E. coli (VTEC). Using the PCR/DNA probe method, 40 strains of VTEC O26 and three strains of VTEC O103 were isolated. IMS-SA identified 21 strains of presumptive E. coli O145, of which only four (19%) were confirmed as serogroup O145. VTEC of this serogroup was not detected by either IMS-SA or PCR/DNA probes. E. coli O111 was not isolated by either method. CONCLUSION: IMS beads were 2.5 times more sensitive than PCR/DNA probe methods for the detection of VTEC O26 in bovine faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: IMS-SA is a sensitive method for detecting specific E. coli serogroups. However, the specificity of this method would be enhanced by the introduction of selective media and the use of tube agglutination tests for confirmation of the preliminary SA results.

Subtyping intimin genes from enteropathogenic Escherichia coli associated with outbreaks and sporadic cases in the United Kingdom and Eire.

PCR-RFLP methods for subtyping the intimin gene from strains of typical and atypical enteropathogenic Escherichia coli (EPEC) and Verocytotoxin-producing E. coli (VTEC) were compared. A novel HhaI PCR-RFLP method was developed that was rapid, easy to use and amplified an 1852 bp fragment of the intimin gene from all isolates examined. This method was used to investigate the intimin sub-types of EPEC strains associated with 14 outbreaks of diarrhoeal disease between 1967 and 2001, and 20 sporadic cases between January and December 2000, in the UK and Eire. In this study, genes encoding alpha, beta, gamma, delta and zeta-intimin were detected in the EPEC strains associated with outbreaks and beta, gamma, epsilon, theta and zeta-intimin genes were identified in isolates from sporadic cases. The beta-intimin gene was the most frequently detected sub-type in both the outbreak and sporadic strains.

An eight-month study of a population of verocytotoxigenic Escherichia coli (VTEC) in a Scottish cattle herd.

AIMS: Strains of Verocytotoxin-producing Escherichia coli (VTEC) from Scottish beef cattle on the same farm were isolated during four visits over a period of eight months. Characteristics of these strains were examined to allow comparisons with strains of VTEC associated with human infection. METHODS AND RESULTS: Strains were characterized to investigate the relationship between these bovine isolates with respect to serotype, Verocytotoxin (VT) type, intimin-type, and presence or absence of the enterohaemolysin genes. VT genes were detected in 176 of 710 (25%) faecal samples tested using PCR, although only 94 (13%) VTEC strains were isolated using DNA probes on cultures. Forty-five different serotypes were detected. Commonly isolated serotypes included O128ab:H8, O26:H11 and O113:H21. VTEC O26:H11 and O113:H21 have been associated with human disease. Strains harbouring the VT2 genes were most frequently isolated during the first three visits to the farm and those with both VT1 and VT2 genes were the major type during the final visit. Of the 94 strains of non-O157 VTEC isolated, 16 (17%) had the intimin gene; nine had the gene encoding beta-intimin and seven strains had an eta/zeta-intimin gene. Forty-one (44%) of 94 strains carried enterohaemolysin genes. CONCLUSIONS: Different serotypes and certain transmissible characteristics, such as VT-type and the enterohaemolysin phenotype, appeared to be common throughout the VTEC population at different times. SIGNIFICANCE AND IMPACT OF THE STUDY: Detailed typing and subtyping strains of VTEC as described in this study may improve our understanding of the relationship between bovine VTEC and those found in the human population.

A mutant of Burkholderia pseudomallei, auxotrophic in the branched chain amino acid biosynthetic pathway, is attenuated and protective in a murine model of melioidosis.

Using a transposon mutagenesis approach, we have identified a mutant of Burkholderia pseudomallei that is auxotrophic for branched chain amino acids. The transposon was shown to have interrupted the ilvI gene encoding the large subunit of the acetolactate synthase enzyme. Compared to the wild type, this mutant was significantly attenuated in a murine model of disease. Mice inoculated intraperitoneally with the auxotrophic mutant, 35 days prior to challenge, were protected against a challenge dose of 6,000 median lethal doses of wild-type B. pseudomallei.

Intimin type influences the site of human intestinal mucosal colonisation by enterohaemorrhagic Escherichia coli O157:H7.

BACKGROUND: Enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli epithelial cell adhesion is characterised by intimate attachment, and attaching and effacing (A/E) lesion formation. This event is mediated in part by intimin binding to another bacterial protein, Tir (translocated intimin receptor), which is exported by the bacteria and integrated into the host cell plasma membrane. Importantly, EPEC (O127:H6) and EHEC (O157:H7) express antigenically distinct intimin types known as intimin alpha and gamma, respectively. EHEC (O157:H7) colonises human intestinal explants although adhesion is restricted to the follicle associated epithelium of Peyer's patches. This phenotype is also observed with EPEC O127:H6 engineered to express EHEC intimin gamma. AIMS: To investigate the influence of intimin on colonisation of human intestine by E coli O157:H7, and intimin types on tissue tropism in humans. METHODS: Human intestinal in vitro organ culture with wild type and mutant strains of O157:H7 were employed. RESULTS: Introducing a deletion mutation in the eae gene encoding intimin gamma in EHEC (O157:H7) caused the strain (ICC170) to fail to colonise human intestinal explants. However, colonisation of Peyer's patches and A/E lesion formation were restored with intimin gamma expression from a plasmid (ICC170 (pICC55)). In contrast, complementing the mutation with intimin alpha resulted in a strain (ICC170 (pCVD438)) capable of colonising and producing A/E lesions on both Peyer's patch and other small intestinal explants. CONCLUSION: Intimin is necessary for human intestinal mucosal colonisation by E coli O157:H7. Intimin type influences the site of colonisation in a Tir type independent mechanism; intimin gamma appears to restrict colonisation to human follicle associated epithelium.

Genome sequence of Yersinia pestis, the causative agent of plague.

The Gram-negative bacterium Yersinia pestis is the causative agent of the systemic invasive infectious disease classically referred to as plague, and has been responsible for three human pandemics: the Justinian plague (sixth to eighth centuries), the Black Death (fourteenth to nineteenth centuries) and modern plague (nineteenth century to the present day). The recent identification of strains resistant to multiple drugs and the potential use of Y. pestis as an agent of biological warfare mean that plague still poses a threat to human health. Here we report the complete genome sequence of Y. pestis strain CO92, consisting of a 4.65-megabase (Mb) chromosome and three plasmids of 96.2 kilobases (kb), 70.3 kb and 9.6 kb. The genome is unusually rich in insertion sequences and displays anomalies in GC base-composition bias, indicating frequent intragenomic recombination. Many genes seem to have been acquired from other bacteria and viruses (including adhesins, secretion systems and insecticidal toxins). The genome contains around 150 pseudogenes, many of which are remnants of a redundant enteropathogenic lifestyle. The evidence of ongoing genome fluidity, expansion and decay suggests Y. pestis is a pathogen that has undergone large-scale genetic flux and provides a unique insight into the ways in which new and highly virulent pathogens evolve.