Ronald A. Dixon

In vitro antibiotic susceptibility of Vibrio parahaemolyticus from environmental sources in northern England

Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium found in estuarine environments worldwide that can be isolated from seawater, sediments, fish (including shellfish) and zooplankton. Gastrointestinal infections are frequently associated with consumption of raw or improperly cooked and contaminated shellfish; less commonly, severe wound infections may occur after exposure of open wounds to contaminated seafood and/or seawater. Antimicrobial agents are used in the treatment and control of prolonged and severe V. parahaemolyticus infection, especially when it occurs in infants, the elderly or immunosuppressed patients. The majority of susceptibility patterns from different geographical regions have previously focused on tropical/subtropical areas in Asia, America and parts of southern Europe, however the present study reports from Northern Europe. The study location is Cleethorpes, Lincolnshire, UK, a recreational resort situated along the River Humber estuary that receives a large number of visitors over the summer months, with several shellfish harvesting activities and mussel cultivation along the coastal line. This study sought to update the antimicrobial resistance status of recent environmental V. parahaemolyticus isolates from this area.

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.

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.

Announcement of population data: genetic data for 17 Y-STR AmpFℓSTR® Yfiler™ markers from an immigrant Pakistani population in the UK (British Pakistanis).

We have analysed 17 Y-chromosomal STR markers (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS438, DYS439, DYS437, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4) for a British Pakistani population. According to the 2010 census, there are more than 1.2 million British Pakistanis in the UK. Substantial numbers of Pakistani immigrants arrived in the UK in the 1950s and 60s and settled mostly in Yorkshire, Lancashire, and in cities such as Birmingham, Bradford, Manchester, Newcastle-on-Tyne and Glasgow in Scotland [1]. By 2001, British Pakistanis were mostly concentrated in the North, West Midlands and London [1] and [2]. Pakistani immigrants came as students to study in British universities, skilled medical doctors to fill medical professional shortage in NHS and as factory workers due to labour shortages in the UK [1]. This population has adopted the life and culture of the UK, however, consanguinity is high [3] and [4].

Genetic variation comparison of 15 autosomal STR loci in an immigrant population living in the UK (British Pakistanis) with an ancestral origin population from Pakistan

n this study, 15 autosomal STR loci were analysed using the AmpFlSTR® Identifiler PCR amplification kit in an immigrant population in the UK (British Pakistanis) and an ancestral origin population (Pakistanis) to elucidate whether an immigrant population can be distinguished from an ancestral origin population from Pakistan.

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.

A Novel in vivo Model of Anaerobic Infection: The Investigation of Clostridium perfringens in Galleria mellonella Larvae.

ABSTRACT Important research progress into the mechanisms of Clostridium perfringens associated diseases (CPAD) has been slowed by the lack of a reliable infection model. Wax moth larvae (Galleria mellonella) have emerged as a viable alternative to traditional mammalian organisms since they are economic, survive at 37°C and require no specialist equipment. This study aims to establish whether G. mellonella larvae can be developed as a viable model for the study of CPAD and their suitability for studying novel treatment strategies. In addition, the study demonstrates a novel time-lapse approach to data collection. Mortality and morbidity rates of larvae challenged with 105 CFU of C. perfringens isolates from various sources were observed over 72h and dose response data obtained using inoculum sizes of 10 - 105 CFU. Phenoloxidase enzyme activity was investigated as a marker for immune response and tissue burden by histopathological techniques. Results show that C. perfringens is pathogenic towards G. mellonella although potency varies between isolates. Infection activates the melanisation pathway resulting in melanin deposition but no increase in enzyme activity was observed. Efficacy of antibiotic therapy (penicillin G, bacitracin, neomycin and tetracycline) administered parenterally loosely correlates with that of in vitro analysis. The findings suggest G. mellonella can be a useful in vivo model of infection when investigating CPAD. Although they are unlikely to replace traditional mammals they may be useful as a pre-screening assay for virulence of C.perfringens strains or as a simple, cheap and rapid in vivo assay in the development and pre-clinical development of novel therapeutics. Highlights Novel in vivo model for the study of Clostridium perfringens infection. Novel time-lapse approach to data collection. First report of the use of G. mellonella model for characterizing virulence in C. perfringens strains. Antibiotic therapy in the model that loosely correlates with in vitro testing.

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).

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.