Mark D. Fielder

Antibodies to Klebsiella, Proteus, and HLA-B27 peptides in Japanese patients with ankylosing spondylitis and rheumatoid arthritis

OBJECTIVE To determine whether patients with ankylosing spondylitis (AS) and patients with rheumatoid arthritis (RA) from Japan have antibodies to Klebsiella pneumoniae and Proteus mirabilis and to assess whether such antibodies are activated against peptides sharing sequences with HLA-B27. METHODS Serum samples from 152 Japanese patients, 52 with AS, 50 with RA, and 50 healthy controls, were tested against 3 bacteria (K. pneumoniae, P. mirabilis, and Escherichia coli) and 3 synthetic peptides (HLA-B27, pullulanase-D, and scrambled pullulanase-D control peptide) by ELISA under coded conditions. Samples were tested for elevations in IgG, IgA, and IgM antibody classes in patients with active AS or RA, in patients with RA with probable disease, and in patients with inactive AS. Disease activity was determined by an elevated serum C-reactive protein (> 10 mg/l) level and elevated erythrocyte sedimentation rate (> 20 mm/h). RESULTS Patients with active AS showed specific elevations in serum IgA antibody levels against K. pneumoniae compared to patients with RA and controls (p < 0.001). No such elevation was seen in the IgG and IgM antibody classes. Patients with inactive AS showed no elevation in any class of antibody against K. pneumoniae compared to controls or patients with RA. Patients with active or probably active RA showed significant elevations in IgG antibody levels against P. mirabilis compared to AS and controls (p < 0.001). Patients with AS (active or inactive), RA (active or probably active), and controls showed no elevations in any antibody class to E. coli. Both active and inactive AS patients had specific autoantibodies against HLA-B27 peptide compared to patients with RA and controls (active AS: IgG, IgA, IgM, p < 0.001; inactive AS: IgG and IgA, p < 0.001). Patients with active AS had IgG and IgA antibodies against pullulanase-D peptide, which contains a sequence that cross reacts with HLA-B27 compared to controls (p < 0.001). CONCLUSION These results provide the first evidence of AS and RA patients in Japan having specific elevations of antibody to K. pneumoniae and P. mirabilis, respectively. This suggests that K. pneumoniae in AS and P. mirabilis in RA may play a role in triggering and/or exacerbating these diseases.

Antimicrobial activities of pomegranate rind extracts: enhancement by addition of metal salts and vitamin C

BackgroundPunica granatum L. or pomegranates, have been reported to have antimicrobial activity against a range of Gram positive and negative bacteria. Pomegranate formulations containing ferrous salts have enhanced although short-term, antibacteriophage activities which are rapidly diminished owing to instability of the ferrous combination. The aim of this study was to determine the antimicrobial activities of combinations of pomegranate rind extracts (PRE) with a range of metals salts with the added stabiliser vitamin C.MethodsPRE solutions, prepared by blending rind sections with distilled water prior to sterilisation by autoclaving or filtration, were screened with a disc diffusion assay using penicillin G as a control. Suspension assays were used to determine the antimicrobial activities of PRE alone and in combination with salts of the following metals; Fe (II), Cu (II), Mn (II) or Zn (II), and vitamin C, against a panel of microbes following exposure for 30 mins. The test organisms included Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis.ResultsThe screening assay demonstrated that PRE exhibited activity against the Gram positive organisms at 24 h with no observable effect on any of the Gram negative bacteria. However, after 12 h, zones of inhibition were only observed for Ps. aeruginosa. In contrast, using the suspension assay, addition of Cu (II) salts to PRE solutions extended the activities resulting in no detectable growth being observed for the PRE/Cu (II) combination against E. coli, Ps. aeruginosa and P. mirabilis. Minimal antimicrobial activity was observed following incubation with Fe (II), Mn (II) or Zn (II) salts alone or in combination with PRE against any of the organisms in the test panel. The addition of vitamin C markedly enhanced the activities of both PRE/Fe (II) and PRE/Cu (II) combinations against S. aureus.ConclusionThis is the first report demonstrating the enhanced efficacy of PRE/metal salt combinations in the presence of the stabilising agent vitamin C, to which all isolates were sensitive with the exception of B. subtilis. This study has validated the exploration of PRE along with additives such as metal salts and vitamin C as novel antimicrobial combinations.

Comparative Genotypic and Phenotypic Characterisation of Methicillin-Resistant Staphylococcus aureus ST398 Isolated from Animals and Humans

The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) ST398 among pigs in certain European countries and North America and its occurrence in other animal species raises a question concerning the molecular mechanisms mediating the success of this lineage. In this study a panel of S. aureus strains belonging to sequence type (ST) 5 (n = 4), ST8 (n = 5), ST15 (n = 5), ST22 (n = 8), clonal complex (CC) 30 (n = 8), CC97 (n = 8), CC130 (n = 4), CC151 (n = 4) and ST398 (n = 18) were screened by DNA microarray and PCR for the carriage of virulence and antimicrobial resistance genes. Isolates belonging to the same sequence type/clonal complex (ST/CC) were found to share similar virulence gene profiles. The ST398 lineage displayed the lowest content of virulence genes, which consisted mainly of genes detected among the majority or all of the analysed lineages. All MRSA ST398 isolates lacked accessory virulence genes that were detected in other ST/CC. In contrast to virulence genotype, the antimicrobial resistance genes profiles varied between isolates belonging to the same ST/CC and profile similarities could be observed for isolates from different lineages. MRSA ST398 isolates in particular displayed significant diversity and high content of antimicrobial resistance genes. This was comparable with certain MRSA belonging to other sequence types particularly the equine MRSA ST8. The apparent lack of significant virulence genes among MRSA ST398 strains, demonstrates that the lineage features a unique genetic background but no ST398-specific virulence markers could be identified.

The antimicrobial properties of copper surfaces against a range of important nosocomial pathogens

Hospital acquired infections (HAI) are a major problem worldwide and controlling the spread of these infections within a hospital is a constant challenge. Recent studies have highlighted the antimicrobial properties of copper and its alloys against a range of different bacteria. The objective of this study was to evaluate the antimicrobial properties of copper compared to stainless steel against a range of clinically important pathogens. These pathogens consisted of five isolates of each of the following organisms; meticillin resistantStaphylococcus aureus (MRSA),Pseudomonas aeruginosa, Escherichia coli, vancomycin-resistant Enterococci (VRE) and Panton-Valentine Leukocidin positive community acquired-MSSA (PVL positive CA-MSSA). MRSA,P. aeruginosa, E. coli, and CA-MSSA isolates were not detectable after a median time of 60 minutes. No detectable levels for all VRE isolates were determined after a median time of 40 minutes. However, for all isolates tested the stainless steel had no effect on the survival of the bacteria and levels remained similar to the time zero count. The results of this study demonstrate that copper has a strong antimicrobial effect against a range of clinically important pathogens compared to stainless steel and potentially could be employed to aid the control HAI.

Cattle, weather and water: mapping Escherichia coli O157:H7 infections in humans in England and Scotland

Entero-haemorrhagic Escherichia coli O157:H7 is a zoonotic pathogen, responsible for a relatively small number of food poisoning and illness outbreaks each year, when compared with other food-borne bacteria capable of causing infections in the population. Nevertheless, E. coli O157:H7 is a bacterial pathogen associated with severe human illnesses including bloody diarrhoea and haemolytic uremic syndrome occurring in both outbreak and sporadic settings. In England and Wales approximately 1% of all laboratory-confirmed cases of food poisoning are the result of E. coli O157:H7; however, in Scotland this figure increases to 3%. When the size of the population is taken into account and the rate of E. coli O157:H7 confirmed cases per 100,000 population is examined, the rate of E. coli 0157:H7 infections in Scotland is much greater than England and Wales. The routes of transmission have changed over time, with new routes of transmission such as farm visits emerging. The prevalence of E. coli O157:H7 has a seasonal dependency, with greater faecal shedding of the organism in the warmer months; this is directly mirrored in the increased reporting of E. coli O157:H7 infection among hospitalized patients. This review attempts to suggest why this phenomenon occurs, paying particular attention to weather, animal movement and private water supplies.

Anti-microbial activities of pomegranate rind extracts: enhancement by cupric sulphate against clinical isolates of S. aureus, MRSA and PVL positive CA-MSSA

BackgroundRecently, natural products have been evaluated as sources of antimicrobial agents with efficacies against a variety of micro-organisms.MethodsThis report describes the antimicrobial activities of pomegranate rind extract (PRE) singularly and in combination with cupric sulphate against methicillin-sensitive and -resistant Staphylococcus aureus (MSSA, MRSA respectively), and Panton-Valentine Leukocidin positive community acquired MSSA (PVL positive CA-MSSA).ResultsPRE alone showed limited efficacy against MRSA and MSSA strains. Exposure to copper (II) ions alone for 2 hours resulted in moderate activity of between 102 to 103 log10 cfu mL-1 reduction in growth. This was enhanced by the addition of PRE to 104 log10 cfu mL-1 reduction in growth being observed in 80% of the isolates. However, the PVL positive CA-MSSA strains were more sensitive to copper (II) ions which exhibited moderate activities of between 103 log10 cfu mL-1 reduction in growth for 60% of the isolates.ConclusionPRE, in combination with Cu(II) ions, was seen to exhibit moderate antimicrobial effects against clinical isolates of MSSA, MRSA and PVL positive CA-MSSA isolates. The results of this study indicate that further investigation into the active ingredients of natural products, their mode of action and potential synergism with other antimicrobial agents is warranted. This is the first report of the efficacy of pomegranate against clinical PVL positive CA-MSSA isolates.

The application of high resolution diffusion NMR to the analysis of manuka honey

The application of DOSY (Diffusion Ordered SpectroscopY) NMR as a technique for the virtual separation of key components of manuka honey and the implications for future discriminatory analysis of honey types is reported for the first time. The scope and the limitations of DOSY NMR are considered using the recently conceived DOSY Tool Box processing software and preliminary anti-bacterial data for the different honey types is reported.

High throughput phenotypic analysis of Mycobacterium tuberculosis and Mycobacterium bovis strains' metabolism using biolog phenotype microarrays.

Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria and already has generated interesting data worthy of further investigation.

Carriage of Staphylococcus species in the veterinary visiting dog population in mainland UK: Molecular characterisation of resistance and virulence

This study investigated the prevalence of nasal carriage of staphylococci in dogs and determined the characteristics of the isolates. A total of 724 dogs from 87 veterinary practices across the mainland UK were screened for carriage of Staphylococcus spp. All isolates were examined for meticillin resistance (MR) and the presence of the mecA gene investigated in those isolates showing resistance. All coagulase-positive staphylococci and MR coagulase-negative staphylococci (MRCoNS) were subjected to antimicrobial susceptibility testing. Spa typing and DNA microarray analysis of resistance and virulence genes was carried out on all MR S. aureus (MRSA) and a subset of meticillin susceptible S. aureus (MSSA). Staphylococci were isolated from 399 (55.1%) of the dogs; only seven (1%) carried MRSA, all of which were identified as the dominant UK healthcare-associated strain (EMRSA-15, ST22). MSSA was identified in 47 (6.5%) dogs, the sequence types of which have been suggested as precursors to successful MRSA clones. Forty (5.5%) dogs carried MRCoNS, while no dogs carried MR S. pseudintermedius, although this is increasingly reported in mainland Europe. Resistance to antimicrobials among the isolates varied between species, with multidrug resistance (MDR) in 87.5% of MRCoNS and 21.8% of coagulase positive staphylococci. Microarray analysis of MRSA and a subset of MSSA isolates identified numerous virulence genes associated with pathogenesis, which are commonly identified in isolates of human origin. However, no isolates carried Panton-Valentine leukocidin (PVL) genes. This study suggests that MRSA carriage is low in the vet visiting dog population, but there is a diverse range of virulence and resistance determinants in canine S. aureus and MRCoNS isolates.

Molecular Mimicry: The Geographical Distribution of Immune Responses to Klebsiella in Ankylosing Spondylitis and its Relevance to Therapy

SummaryThe discovery that HLA- B27 is linked to ankylosing spondylitis (AS) and HLA- DR1/DR4 to rheumatoid arthritis (RA) has provided new approaches to the study of the possible causation of these diseases. Several theories have been proposed to explain these associations but only one, namely “molecular mimicry”, has provided a specific aetiological agent for each of these diseases. p]Molecular mimicry between HLA- B27 and two molecules in Klebsiella microbes: nitrogenase and pullulanase D has been reported whilst in Proteus microbes, the haemolysin molecule shows sterochemical similarity to HLA- DR1/DR4. Elevated immune responses to Klebsiella microbes have been demonstrated in AS patients from 10 different countries and this wide geographical distribution suggests that the same aetiological agent is probably acting in producing this condition. p]Furthermore RA patients show similar immune responses to Proteus microbes. Whether AS or RA are caused by these bacteria can only be resolved by tissue typing all rheumatol ogical patients early, in the course of their disease and then assessing their response to antibiotic chemotherapy in longitudinal studies involving double- blind crossover trials. p]It is possible that in the future, the course of AS or even RA could be modified by adequate antibiotic chemotherapy or even diets which affect the substrates on which these bacteria grow.