A Critical Review of AMR Risks Arising as a Consequence of Using Biocides and Certain Metals in Food Animal Production.

The focus of this review was to assess what evidence exists on whether, and to what extent, the use of biocides (disinfectants and sanitizers) and certain metals (used in feed and other uses) in animal production (both land and aquatic) leads to the development and spread of AMR within the food chain. A comprehensive literature search identified 3434 publications, which after screening were reduced to 154 relevant publications from which some data were extracted to address the focus of the review. The review has shown that there is some evidence that biocides and metals used in food animal production may have an impact on the development of AMR. There is clear evidence that metals used in food animal production will persist, accumulate, and may impact on the development of AMR in primary animal and food production environments for many years. There is less evidence on the persistence and impact of biocides. There is also particularly little, if any, data on the impact of biocides/metal use in aquaculture on AMR. Although it is recognized that AMR from food animal production is a risk to human health there is not sufficient evidence to undertake an assessment of the impact of biocide or metal use on this risk and further focused in-field studies are needed provide the evidence required.

A metagenomic analysis of the bacterial microbiome of limestone, and the role of associated biofilms in the biodeterioration of heritage stone surfaces.

There is growing concern surrounding the aesthetic and physical effects of microbial biofilms on heritage buildings and monuments. Carboniferous stones, such as limestone and marble, are soluble in weak acid solutions and therefore particularly vulnerable to biocorrosion. This paper aims to determine the differences and commonalities between the microbiome of physically damaged and undamaged Lincolnshire limestone, an area of research which has not been previously studied. A lack of information about the core microbiome has resulted in conflicting claims in the literature regarding the biodeteriorative potential of many microorganisms. To address this, we used metagenomics alongside traditional microbiological techniques to produce an in-depth analysis of differences between the bacterial microbiomes found on deteriorated and undamaged external limestone surfaces. We demonstrate there is a core microbiome on Lincolnshire limestone present on both damaged and undamaged surfaces. In addition to the core microbiome, significant differences were found between species isolated from undamaged compared to damaged surfaces. Isolated species were characterised for biofilm formation and biodeteriorative processes, resulting in the association of species with biodeterioration that had not been previously described. Additionally, we have identified a previously undescribed method of biofilm-associated biomechanical damage. This research adds significant new understanding to the field, aiding decision making in conservation of stone surfaces.

Dynamics of toxigenic Clostridium perfringens colonisation in a cohort of prematurely born neonatal infants.

BACKGROUND: Clostridium perfringens forms part of the human gut microbiota and has been associated with life-threatening necrotising enterocolitis (NEC) in premature infants. Whether specific toxigenic strains are responsible is unknown, as is the extent of diversity of strains in healthy premature babies. We investigated the C. perfringens carrier status of premature infants in the neonatal intensive care unit, factors influence this status, and the toxic potential of the strains. METHODS: C. perfringens was isolated by culture from faecal samples from 333 infants and their toxin gene profiles analysed by PCR. A survival analysis was used to identify factors affecting probability of carriage. Competitive growth experiments were used to explore the results of the survival analysis. RESULTS: 29.4% of infants were colonized with C. perfringens before they left hospital. Three factors were inversely associated with probability of carriage: increased duration of maternal milk feeds, CPAP oxygen treatment and antibiotic treatment. C. perfringens grew poorly in breast milk and was significantly outperformed by Bifidobacterium infantis, whether grown together or separately. Toxin gene screening revealed that infants carried isolates positive for collagenase, perfringolysin O, beta 2, beta, becA/B, netB and enterotoxin toxin genes, yet none were observed to be associated with the development of NEC. CONCLUSIONS: Approximately a third of preterm infants are colonised 3 weeks after birth with toxin gene-carrying C. perfringens. We speculate that increased maternal breast milk, oxygen and antibiotic treatment creates an environment in the gut hostile to growth of C. perfringens. Whilst potentially toxigenic C. perfringens isolates were frequent, no toxin type was associated with NEC. TRIAL REGISTRATION: clinicaltrials.gov NCT01102738, registered 13th April 2010.

Genomic analysis on broiler-associated Clostridium perfringens strains and exploratory caecal microbiome investigation reveals key factors linked to poultry necrotic enteritis.

BACKGROUND: Clostridium perfringens is a key pathogen in poultry-associated necrotic enteritis (NE). To date there are limited Whole Genome Sequencing based studies describing broiler-associated C. perfringens in healthy and diseased birds. Moreover, changes in the caecal microbiome during NE is currently not well characterised. Thus, the aim of this present study was to investigate C. perfringens virulence factors linked to health and diseased chickens, including identifying putative caecal microbiota signatures associated with NE. RESULTS: We analysed 88 broiler chicken C. perfringens genomes (representing 66 publicly available genomes and 22 newly sequenced genomes) using different phylogenomics approaches and identified a potential hypervirulent and globally-distributed clone spanning 20-year time-frame (1993-2013). These isolates harbored a greater number of virulence genes (including toxin and collagen adhesin genes) when compared to other isolates. Further genomic analysis indicated exclusive and overabundant presence of important NE-linked toxin genes including netB and tpeL in NE-associated broiler isolates. Secondary virulence genes including pfoA, cpb2, and collagen adhesin genes cna, cnaA and cnaD were also enriched in the NE-linked C. perfringens genomes. Moreover, an environmental isolate obtained from farm animal feeds was found to encode netB, suggesting potential reservoirs of NetB-positive C. perfringens strains (toxinotype G). We also analysed caecal samples from a small sub-set of 11 diseased and healthy broilers for exploratory microbiome investigation using 16S rRNA amplicon sequencing, which indicated a significant and positive correlation in genus Clostridium within the wider microbiota of those broilers diagnosed with NE, alongside reductions in beneficial microbiota members. CONCLUSIONS: These data indicate a positive association of virulence genes including netB, pfoA, cpb2, tpeL and cna variants linked to NE-linked isolates. Potential global dissemination of specific hypervirulent lineage, coupled with distinctive microbiome profiles, highlights the need for further investigations, which will require a large worldwide sample collection from healthy and NE-associated birds.

Prospects for Biocontrol of Vibrio parahaemolyticus Contamination in Blue Mussels (Mytilus edulus)-A Year-Long Study.

Vibrio parahaemolyticus is an environmental organism normally found in subtropical estuarine environments which can cause seafood-related human infections. Clinical disease is associated with diagnostic presence of tdh and/or trh virulence genes and identification of these genes in our preliminary isolates from retail shellfish prompted a year-long surveillance of isolates from a temperate estuary in the north of England. The microbial and environmental analysis of 117 samples of mussels, seawater or sediment showed the presence of V. parahaemolyticus from mussels (100%) at all time-points throughout the year including the colder months although they were only recovered from 94.9% of seawater and 92.3% of sediment samples. Throughout the surveillance, 96 isolates were subjected to specific PCR for virulence genes and none tested positive for either. The common understanding that consuming poorly cooked mussels only represents a risk of infection during summer vacations therefore is challenged. Further investigations with V. parahaemolyticus using RAPD-PCR cluster analysis showed a genetically diverse population. There was no distinct clustering for "environmental" or "clinical" reference strains although a wide variability and heterogeneity agreed with other reports. Continued surveillance of isolates to allay public health risks are justified since geographical distribution and composition of V. parahaemolyticus varies with Future Ocean warming and the potential of environmental strains to acquire virulence genes from pathogenic isolates. The prospects for intervention by phage-mediated biocontrol to reduce or eradicate V. parahaemolyticus in mussels was also investigated. Bacteriophages isolated from enriched samples collected from the river Humber were assessed for their ability to inhibit the growth of V. parahaemolyticus strains in-vitro and in-vivo (with live mussels). V. parahaemolyticus were significantly reduced in-vitro, by an average of 1 log-2 log units and in-vivo, significant reduction of the organisms in mussels occurred in three replicate experimental tank set ups with a "phage cocktail" containing 12 different phages. Our perspective biocontrol study suggests that a cocktail of specific phages targeted against strains of V. parahaemolyticus provides good evidence in an experimental setting of the valuable potential of phage as a decontamination agent in natural or industrial mussel processing (343w).

Molecular screening of blue mussels indicated high mid-summer prevalence of human genogroup II Noroviruses, including the pandemic "GII. 4 2012" variants in UK coastal waters during 2013

Abstract This molecular study is the first report, to the best of our knowledge, on identification of norovirus, NoV GII.4 Sydney 2012 variants, from blue mussels collected from UK coastal waters. Blue mussels (three pooled samples from twelve mussels) collected during the 2013 summer months from UK coastal sites were screened by RT-PCR assays. PCR products of RdRP gene for noroviruses were purified, sequenced and subjected to phylogenetic analysis. All the samples tested positive for NoVs. Sequencing revealed that the NoV partial RdRP gene sequences from two pooled samples clustered with the pandemic "GII.4 Sydney variants" whilst the other pooled sample clustered with the NoV GII.2 variants. This molecular study indicated mussel contamination with pathogenic NoVs even during mid-summer in UK coastal waters which posed potential risk of NoV outbreaks irrespective of season. As the detection of Sydney 2012 NoV from our preliminary study of natural coastal mussels interestingly corroborated with NoV outbreaks in nearby areas during the same period, it emphasizes the importance of environmental surveillance work for forecast of high risk zones of NoV outbreaks.

Molecular screening of blue mussels indicated high mid-summer prevalence of human genogroup II Noroviruses, including the pandemic "GII.4 2012" variants in UK coastal waters during 2013.

This molecular study is the first report, to the best of our knowledge, on identification of norovirus, NoV GII.4 Sydney 2012 variants, from blue mussels collected from UK coastal waters. Blue mussels (three pooled samples from twelve mussels) collected during the 2013 summer months from UK coastal sites were screened by RT-PCR assays. PCR products of RdRP gene for noroviruses were purified, sequenced and subjected to phylogenetic analysis. All the samples tested positive for NoVs. Sequencing revealed that the NoV partial RdRP gene sequences from two pooled samples clustered with the pandemic "GII.4 Sydney variants" whilst the other pooled sample clustered with the NoV GII.2 variants. This molecular study indicated mussel contamination with pathogenic NoVs even during mid-summer in UK coastal waters which posed potential risk of NoV outbreaks irrespective of season. As the detection of Sydney 2012 NoV from our preliminary study of natural coastal mussels interestingly corroborated with NoV outbreaks in nearby areas during the same period, it emphasizes the importance of environmental surveillance work for forecast of high risk zones of NoV outbreaks.

Non-antibiotic Isotretinoin Treatment Differentially Controls Propionibacterium acnes on Skin of Acne Patients.

Emergence and potential transfer of antibiotic resistance in skin microorganisms is of current concern in medicine especially in dermatology contexts where long term treatment with antibiotics is common. Remarkably, non-antibiotic therapy in the form of isotretinoin - a non-antimicrobial retinoid is effective at reducing or eradicating the anaerobe Propionibacterium acnes which is causally involved in the complex pathogenesis of Acne vulgaris. This study measured the extent of colonization of P. acnes in patients with primary cystic or severe acne from three defined skin sites in 'non-lesion' areas before, during and after treatment with isotretinoin. Patients attending acne clinics were investigated using standardized skin sampling techniques and the recovery of anaerobic P. acnes from 56 patients comprising 24 females and 32 males (mean age 22 years, age range 15-46 years) who were given a standard course of isotretinoin (1 mg/kg/day) are reported. P. acnes cultured from the external cheek surface of patients following treatment showed a significant reduction (1-2 orders of magnitude) compared with their pre-treatment status. Interestingly, other distinct sites (nares and toe web) failed to show this reduction. In addition, high levels of antibiotic-resistant P. acnes were recorded in each patients' skin microbiota before, during and after treatment. In this study, microbial composition of the skin appears substantially altered by isotretinoin treatment, which clearly has differential antimicrobial effects on each anatomically distinct site. Our study confirmed that orally administered isotretinoin shows good efficacy in the resolution of moderate to severe acne that correlates with reductions in the number of P. acnes on the skin, including resistant isolates potentially acquired from previous treatments with antibiotics. Our study suggests that the role of tetracycline's and macrolides, which are currently first line treatments in dermatology, might be reserved for severe or life-threatening infections since current antibiotic stewardship guidelines from medical departments no longer prescribe these antibiotics for routine use.

The identification of proteoglycans and glycosaminoglycans in archaeological human bones and teeth.

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.

DNA and bone structure preservation in medieval human skeletons.

Morphological and ultrastructural data from archaeological human bones are scarce, particularly data that have been correlated with information on the preservation of molecules such as DNA. Here we examine the bone structure of macroscopically well-preserved medieval human skeletons by transmission electron microscopy and immunohistochemistry, and the quantity and quality of DNA extracted from these skeletons. DNA technology has been increasingly used for analyzing physical evidence in archaeological forensics; however, the isolation of ancient DNA is difficult since it is highly degraded, extraction yields are low and the co-extraction of PCR inhibitors is a problem. We adapted and optimised a method that is frequently used for isolating DNA from modern samples, Chelex(®) 100 (Bio-Rad) extraction, for isolating DNA from archaeological human bones and teeth. The isolated DNA was analysed by real-time PCR using primers targeting the sex determining region on the Y chromosome (SRY) and STR typing using the AmpFlSTR(®) Identifiler PCR Amplification kit. Our results clearly show the preservation of bone matrix in medieval bones and the presence of intact osteocytes with well preserved encapsulated nuclei. In addition, we show how effective Chelex(®) 100 is for isolating ancient DNA from archaeological bones and teeth. This optimised method is suitable for STR typing using kits aimed specifically at degraded and difficult DNA templates since amplicons of up to 250bp were successfully amplified.

The antibacterial potency of the medicinal maggot, Lucilia sericata (Meigen): variation in laboratory evaluation.

Research to quantify the potency of larval excretion/secretion from Lucilia sericata using liquid culture assays has produced contradictory results. In this study, viable counting was used to investigate the effectiveness of excretion/secretion against three marker bacterial species (Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli) and the effects of varying growing conditions in assays. Results demonstrate that factors such as number of larvae, species of bacteria and addition of nutrient influence its antibacterial potency. Therefore a standardised method should be employed for liquid culture assays when investigating the antibacterial activity of larval excretion/secretion from L. sericata.

Role of the cell envelope in the antibacterial activities of polymyxin B and polymyxin B nonapeptide against Escherichia coli.

The role of membrane permeabilisation and disruption in the mechanism of action of some polymyxin analogues against Gram-negative organisms is contentious. The effects of polymyxin B (PMB) and its analogue polymyxin B nonapeptide (PMBN) on Escherichia coli envelopes should correlate, but previous work by other workers suggests that PMBN has a different mode of action. This study has reassessed the biochemical techniques used previously and has shown that, in contrast to previous studies, PMBN (a well-characterised antibacterial synergist) readily releases periplasmic proteins and lipopolysaccharide from treated E. coli at subinhibitory concentrations in normal physiological buffer conditions. We conclude that, when tested with appropriate methodology, PMBN closely correlates with the early effects of PMB on the cell envelope of E. coli and this study shows that it is now consistent with the accepted interactions of membrane-active agents against Gram-negative cells.

Outer membrane protein analysis of ampicillin-resistant isolates of Haemophilus influenzae from Saudi Arabia.

Haemophilus influenzae isolates characterized in a previously published study from the Kingdom of Saudi Arabia were analysed by outer membrane protein (OMP) profiling. Isolates from patients with confirmed respiratory tract infections were investigated. Antibiotic susceptibility tests in vitro showed 25/129 (19.4%) had various degrees of reduced susceptibility to ampicillin although all were fully susceptible to ceftazidime and ciprofloxacin. OMP profiles of the beta-lactamase mediated ampicillin-resistant and beta-lactamase negative; ampicillin intermediate resistant strains (BLNAI) isolated were investigated. Dendrograms of scanned SDS-PAGE of these strains showed 15 different groupings from the 15 non-typable (NTHi) isolates tested demonstrating a high degree of heterogeneity whereas the 5 Hib isolates demonstrated significantly closer relatedness and were probably clonal. The present study demonstrates the groupings of H. influenzae strains by OMP profile analysis which did not correlate with the beta-lactamase production ability, BLNAI isolates, geographical origin or biotype.

Contaminated operating room boots: the potential for infection.

BACKGROUND: Dirty operating room boots, often contaminated with blood and other infected material, are not only a source of discontent among surgeons and other surgical personnel, but they also pose a potential risk of transmission of viral or bacterial diseases to the wearer and cleaner of the boots. METHOD: Operating room boots were examined for the presence of blood by visual inspection; the presence or absence of blood was confirmed by a specific biochemical test. Bacterial isolation and quantification from boots were performed with conventional methodology. RESULTS: In this study, a spot check revealed that 44% of all operating room boots tested were contaminated with blood and that the majority were contaminated with bacteria. Sixty-three percent of surgeons using the facility had blood-contaminated boots, and a significant number of boots belonging to other surgical personnel were also contaminated with blood and bacteria normally associated with skin microbiota or the environment. Comfort shoes with perforations on their upper surface and plastic boots commonly found in operating rooms were most heavily contaminated, whereas Wellington boots and clogs had less contamination. CONCLUSION: The present practice of manual cleaning of boots is unsatisfactory, and it is recommended that boots be washed in automatic washing machines.