Data has emerged which indicates that antimicrobial use in animals has created a reservoir of resistant bacteria and resistance genes that have spread to humans. The aim of this study was to examine the influence of animal antimicrobial use on human strains of S. aureus. Phenotypic and genotypic methods assessed the genetic population structure, potential for host adaptation, frequency of antimicrobial resistance, presence and frequency of genes encoding tetracycline and macrolide resistance and structural variation in tetK genes in S. aureus from animals compared with human clinical strains. In addition, the transferability of tetK resistance plasmids from animal strains to S. aureus 8325-4 was investigated. DNA based typing exhibited 100% typeability, high levels of discrimination and a high degree of concordance between methods. Multi-locus sequence typing (MLST) identified six sequence types (CC5, eC15, CC22, CC25, CC30 and CC45) common to both isolate collections that represented four of the five major human methicillin resistant Staphylococcus aureus (MRSA) clonal lineages. The relatedness of these clones was further supported by the analysis of20 different virulence determinants. Isolates of ee5 exhibited resistance to ciprofloxacin, clindamycin, erythromycin, penicillin, streptomycin, tobramycin and tylosin, CC15 to penicillin and tetracycline, CC22 to penicillin and rifampicin and eC30 to penicillin. Isolates of CC45 were fully susceptible. Low level biocide resistance was detected but the significance of this was unclear. The lelK and ermC genes were the predominant tetracycline and macrolide resistance genes. lelK was harboured by animal isolates of CC5 and CC15 and ermC by animal isolates of CC5. Restriction fragment length polymorphism (RFLP) of lelK amplicons produced indistinguishable restriction patterns. High frequency transfer of a lelK plasmid from a chicken S. aureus (CC5) to S. aureus 8325-4 was observed. These data support the hypothesis that animals represent an important reservoir of antibiotic resistant S. aureus with the ability for strain and antibiotic resistance gene transfer to humans.

No description available.

No description available.

Methicillin resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen and morbidity and mortality rates associated with this pathogen have increased markedly in recent years. The prevalence of MRSA is no longer confined to hospital patients since MRSA infections have been increasingly reported in the community. More recently, community-acquired MRSA strains have become more prevalent and their infections are no longer confined to the community but have started to replace hospital-acquired MRSA in some health care settings. MRSA strains are generally resistant to several classes of antibiotics and are therefore difficult and costly to treat. Consequently, an understanding of the epidemiological characteristics of S. aureus is an essential tool for the management of its infections in both the hospital and community setting. The purpose of any epidemiology study, such as the investigation of an outbreak, is to identify the potential source(s) of an infection and to monitor their dissemination. The early identification of an outbreak, making use of a rapid, precise and simple MRSA typing technique, can lead to prompt and effective precautions that avoid further spread of the infection. Pulsed-field gel electrophoresis (PFGE) is considered the gold standard for MRSA typing and has been recently supported by multi-locus sequence typing (MLST). However, technical limitations restrict the use of PFGE and MLST in the majority of routine hospital laboratories: they are time-consuming, expensive, require specific expertise and specialist equipment. In this study a novel typing technique was developed for S. aureus, based on single nucleotide polymorphism (SNP) variations in and around SmaI-restriction sites (CCCGGG), following the analysis of eighteen S. aureus strains that have had their genomes sequenced. The developed SmaI-multiplex PCR genotyping method combines the high discriminatory power and reproducibility of PFGE, with the simplicity of a multiplex PCR-based technique that can be performed in a routine clinical laboratory. The validity of SmaI-multiplex PCR was carefully assessed in comparison with PFGE and MLST against many sequenced S. aureus strains and showed high discriminatory power and reproducibility. There was also high level of concordance in the clustering of strains analyzed by each of the techniques. The SmaI-multiplex PCR was ultimately evaluated against a large number of clinical S. aureus outbreak strains and was shown to be a useful tool for providing reliable epidemiological information for the investigation of clinical staphylococcal outbreaks. The newly developed technology is suitable for high throughput sample analysis, is relatively cheap and provides reliable and comparable genotyping data. At the same time, the SmaI-multiplex PCR meets most of the criteria of practical typing method: it is simple, inexpensive, highly discriminatory and does not require sophisticated equipment or expertise. Consequently, SmaI-multiplex PCR could be used routinely in any clinical microbiology laboratory since it relies on standard clinical laboratory apparatus (i.e. PCR machine and agarose gel electrophoresis). SmaI-multiplex PCR proved to be more discriminatory than MLST/SCCmec typing, but less discriminatory than PFGE. Currently the SmaI-multiplex PCR protocol takes between 4 to 6 hours; however, it would be possible to adapt this technology towards an automated genotyping assay using RT-multiplex PCR. This would reduce the processing time to less than 60 minutes. Since individual targets are identifiable on the basis of the size of their amplicons, the RT-PCR output could be processed directly via dedicated analytical software.

Research into chronic hepatitis C virus (HCV) infection (CHC) is needed because only ~50% of patients with CHC are cured with existing treatments. HCV interacts with metabolism of cholesterol and very low density lipoproteins (VLDL) for replication, assembly, secretion and entry. Patients with CHC exhibit a dyslipidemia characterised by low LDL cholesterol (LDL-C) and insulin resistance. This translational study characterised the dyslipidemia apparent in CHC in retrospective and prospective HCV cohorts. Distinct metabolic phenotypes were defined between HCV genotypes 1 and 3. LDL-C was markedly reduced in HCV-G3, which exerted a greater effect than apoE genotype on LDL-C levels. Prospective analysis of non-cholesterol sterol intermediates established that disordered cholesterol synthesis in HCV was mediated predominantly via the lathosterol pathway. In HCV-G3, levels of Proprotein Convertase Subtilisin Kexin type 9 (PCSK9) were low compared to healthy controls, suggesting increased LDL clearance. Low LDL-C and high triglyceride/HDL ratio were found to be predictive of poor response to anti-viral therapy. Collaboration in a genome wide association study revealed that SNPs in IL28B rather than in lipid regulating genes are the major host genetic determinants of treatment response. Analysis of HCV lipoviral particles (LVP) by iodixanol density gradient ultracentrifugation revealed correlations between insulin resistance and triglycerides (TG) with LVP in HCV-G1. Determination of apoB in VLDL1, VLDL2, IDL and LDL fractions confirmed fasting TG are predominately in the VLDL1 fraction, implying that HCV-G1 preferentially associates with the VLDL1 pathway. Interferon γ inducible protein 10 (IP10), a marker of hepatic interferon stimulated gene expression correlated with LDL-C and HCV LVP ratio in HCV-G1, explaining the association between low LDL-C and poor treatment response. A randomised pilot trial in 60 CHC non-responders indicated that 12 weeks of treatment with Fluvastatin can lower total viral load in HCV-G1 & G4 and that low dose n3 PUFAs improved IP10 levels.

No description available.

Gastrointestinal helminths infect over 1 billion people worldwide. While rarely causing death, intestinal helminths cause high morbidity, especially in children and result in huge economic losses each year. While antihelminthics have proven effective, in endemic areas reinfection is common and the emergence of antihelminthic resistance is a major concern. The murine intestinal nematode, Trichuris muris has been utilised as a good model for the human parasite, T. trichiura for the past 45 years and has yielded much evidence of the immune response to this caecal-dwelling nematode. Studies of the immune response to T. muris have mainly been conducted using the E isolate. Host resistance to T. muris relies upon the development of a Th2 response while susceptibility correlates with a Th1 response. In the present work, studies were conducted using the J and S isolates and the conventionally-studied E isolate. Firstly, the phylogenetic relationship between the E, J and S isolates was explored by comparing internal transcribed spacer (ITS) 2 sequences between the isolates and to other ITS2 sequences from other Trichuris species. These analyses revealed the isolates to be extremely similar at this level. Following this, the immune response elicited by the E, J and S isolates of T. muris in male AKR, C57BL/6 and BALB/c mice was characterised. In BALB/c mice, all isolates were expelled and was, with all three isolates, associated with an elevated Th2 response. In contrast, all three isolates persisted to chronicity in AKR mice but the E and S isolate elicited significantly higher IL-12 while levels in J-infected mice were equivalent to naïve. Serum MMCP-1 was significantly higher in J and E-infected mice compared with S-infected mice. Interestingly, differential worm expulsion kinetics occurred in the C57BL/6 mouse strain, with the E and J isolate expelled by d 35 p.i. but the S isolate was retained to chronicity. Furthermore, levels of Th2 cytokines were markedly lower in S-infected mice while type 1 cytokines IL-12 and IFN-γ were raised in all infected groups. The lower Th2 response in S-infected mice was reflected in a lower goblet cell hyperplasia and a lower mastocytosis and eosinophilia. IgG1 was equally raised while IgG2a was lowest in J-infected mice. In contrast, female C57BL/6 mice were able to mount a Th2 response against the S isolate and expel most worms by d 35 p.i. Furthermore, survival of the S isolate is dependent upon MyD88 thus MyD88 KO mice expelled the S isolate in a Th2-dependent manner. To begin to address the mechanism underlying S persistence, the effects of isolate-specific E/S on DC were examined. Interestingly, CD 1lc+ DC from C57BL/6 mice upregulated MHC II expression when stimulated with E and J E/S but did not with S E/S. Western blots showed parasite-specific E/S to be differentially recognised by mouse strain-specific antibody, with strong recognition of low molecular weight antigens being associated with susceptibility. Furthermore, E/S zymography revealed protease activity at approximately 20 kDa which was greatest in S isolate E/S. Inhibition studies showed differential protease activity between isolate specific E/S but all exhibited serine protease activity. Overall, these studies enhanced previous understanding of the immune response to T. muris as a whole thus highlighting the relevance parasite isolates have in parasite immunology.

Trichuriasis, caused by the whipworm T. trichiura, is endemic in tropical and subtropical areas, affecting approximately one fifth of the world's population. Child anthelminthic treatment programmes are being implemented in countries with endemic disease but repeated treatments are costly, may prevent the development of acquired immunity and can lead to the development of drug resistant parasites. Thus, the development of a vaccine which would lead to the acquisition of immunity at an earlier age and reduce community faecal egg output would be beneficial. Development of subunit vaccines requires the identification of protective antigens and their formulation in a suitable adjuvant. T. muris is an antigenically similar laboratory model for T. trichiura. Subcutaneous (s.c.) vaccination with adult excretory-secretory (ES) proteins protects susceptible mouse strains from T. muris. Larval stages may contain novel antigens which when incorporated in a vaccine induce worm expulsion earlier in infection. This study finds no difference in the cellular and humoral immune response to T. muris adult and L3 ES but identifies high molecular weight proteins in both adult and L3 ES as potential vaccine candidates. Recombinant IL-13 failed to restore protection to a nonprotective vaccine dose of ES, but further work is required prior to ruling out IL-13 as a potential Th2 polarising adjuvant. This is the first study to describe the cellular and humoral components of the protective immune response following s.c. vaccination against T. muris. Protection of susceptible mice involved the development of a Th2 response and down-regulation of IL-12 while IFN-γ levels remained high. Goblet cell hyperplasia and macrophage influx were induced by vaccination. Epithelial turnover did not appear to be increased by vaccination, suggesting that there are differences in the mechanisms of expulsion between 'natural resistance' and 'vaccinated resistance'. Genes involved in serum IgG1 production were highly expressed in the peripheral lymph node cells (PLNC) following s.c. vaccination with ES/FIA. This, combined with the high levels of serum IgGl and mucosal IgGl and IgA in the colon of mice protected by the ES/FIA vaccine suggests that antibody is involved in vaccination-induced worm expulsion. This is further supported by the fact that anti-CCR10 treatment of PLNC prior to adoptive transfer to SCID mice reduces lymphocyte recruitment to the colonic mucosa and delays worm expulsion. This is the first study on the role of CCL11 in eosinophil recruitment to the small and large intestine during gastrointestinal helminth infection. A peripheral eosinophilia was seen in wild type BALB/c mice and CCL11 deficient mice infected with T spiralis but not with T muris. Gastrointestinal eosinophilia was markedly reduced but not ablated in CCL11 deficient mice - and negligible in CCL11 IL-5 deficient mice - infected with either nematode. The residual eosinophilia and up-regulation of CCL24 mRNA in the gastrointestinal tract of CCL11 deficient mice infected with either nematode, together with the presence of an eosinophilactive factor in T. spiralis and T. muris products, suggest that CCL11 is the salient but not the sole eosinophil chemoattractant of biological significance during gastrointestinal helminth infection.

Cystic fibrosis (CF) patients are predisposed to a number of bacterial infections, including <italic>Pseudomonas aeruginosa</italic> and the <italic>Burkholderia cepacia</italic> complex (Bcc). Both groups of bacteria have been associated with contamination of products containing biocides, leading to concerns that the over use of biocide products could select for multi drug resistant organisms. This investigation examined the susceptibility profile of panels of <italic>P. aeruginosa</italic> and Bcc strains to a range of biocides including chlorhexidine and cetylpyridinium chloride (CPC). It was found that certain epidemic strains that had spread among individuals with CF, such as the <italic>B. cenocepacia</italic> J2315 strain lineage and the <italic>P. aeruginosa</italic> Liverpool strain were less susceptible to chlorhexidine than other strains representative of the same species. Although minimum inhibitory concentration (MIC) screens gave an overall view of biocide susceptibility, minimum bactericidal concentrations (MBC) for these bacteria were higher. For CPC 27 out of 40 strains required more than 20 fold more biocide than the MIC to achieve a bactericidal effect. Suspension tests were performed on two commercial biocide formulations, a chlorhexidine-based wash, Hibiscrub and a triclosan-based hand gel Cuticura . The epidemic <italic> B. cenocepacia</italic> strain J2315 was capable of surviving in both these products after 20 minutes of exposure and viable bacteria were isolated after 60 minutes of exposure in Hibiscrub . The results from this investigation suggest that certain commercial biocides are not effective against the Bcc. Therefore to assist in the future development of biocides, three highly resistant Bcc strains were proposed as suitable reference strains to use in challenge testing for biocide efficacy. The molecular basis of biocide resistance was determined using a microarray approach to profile global gene expression of <italic> B. cenocepacia</italic> in response to chlorhexidine. <italic>B. cenocepacia </italic> J2315 was exposed to sub-inhibitory levels of chlorhexidine (5 ug/ml) and expression compared to cells not exposed to biocide. The microarray analysis demonstrated significant alterations in expression at P < 0.05, with a > 1.5 fold change with 98 up-regulated and 76 down-regulated genes. Two chlorhexidine up-regulated genes were selected for further analysis, a response regulator (BCAM 0924) potentially involved with efflux and a novel transport related gene (BCAL 2553). Site directed mutagenesis of these genes was carried out in <italic>B. cenocepacia</italic> strain K56-2 and a reduction in chlorhexidine MIC was observed for each respective mutant compared to the wildtype. 66 out of 76 (87%) of the down-regulated genes were involved with motility related functions. This led to the hypothesis that sub-inhibitory levels of chlorhexidine inhibited swarming motility and induced biofilm production. This question was tested for Bcc strains using soft- agar swarming tests and 96-well plate biofilm assays. A total of 6 of 10 strains screened exhibited both biofilm induction and swarming inhibition in response to chlorhexidine. A potential conserved regulatory binding motif was observed upstream of all gene sets down- regulated chlorhexidine in the microarray analysis. This suggested that swarming inhibition and biofilm induction in <italic>B. cenocepacia</italic> may be controlled by a coordinated regulatory pathway controlled by a two component system sensor-regulator system. A transposon mutagenesis approach was used to identify <italic>B. cenocepacia</italic> mutants that lacked the inhibitory response. The screen identified several mutations involved in the phenomenon including cheY-Uke receiver genes and a glycosyl transferase encoding gene. In conclusion, the molecular analysis of biocide resistance in Bcc bacteria demonstrated it was multifactoral, involving efflux pumps, transport related genes, membrane proteins and regulatory genes. The ability of chlorhexidine to inhibit swarming and switch Bcc to a non motile biofilm lifestyle was identified as a novel biocide survival response. With further research this regulatory pathway may be a potential target for the development of a novel biocides and therapeutics which overcome the antimicrobial resistance of these bacteria pathogens.

<italic>Staphylococcus aureus</italic> is a major cause of hospital-acquired infections that are becoming increasingly difficult to treat because of the organism's ability to acquire resistance to current antimicrobial agents. Particular attention has been focussed on the evolution of methicillin-resistant <italic> S. aureus</italic> (MRSA) - strains of <italic>S. aureus</italic> that are, in some cases, resistant to almost all known antibiotic classes. One method used to control the spread of MRSA has been the use of topical washes that include triclosan, a potent antimicrobial with particular activity against Gram-positive organisms. Triclosan has traditionally been classed as a biocide and is also used in a broad spectrum of consumer healthcare products, including toothpastes and deodorants. It has also been used to prevent bacterial growth through incorporation into plastics used during food preparation or sutures used to close wounds following surgery. However, in 1991 resistance to triclosan was reported and was described to transfer in association with mupirocin resistance. This was followed by reports that resistance was present in 7.5% of <italic> S. aureus</italic> isolates and that MRSA was less susceptible to triclosan than methicillin- sensitive <italic>S. aureus</italic> (MSSA). It later emerged that, contrary to previous thinking, triclosan targets a specific bacterial protein, Fabl. We aimed to characterize the development of reduced susceptibility to triclosan in MSSA and MRSA and to identify whether triclosan does have a single, specific target. We also set out to elucidate the potential for triclosan resistance to be disseminated by horizontal gene transfer (HGT). By using extensive microbiological and genetic techniques we found that <italic> S. aureus</italic> can evolve reduced susceptibility to triclosan through spontaneous mutation. MICs of 1-4 mg/L were achieved by a C284T mutation offabl, compared to wild-type MICs of -0.03 mg/L. However, reduced susceptibility was also observed in non-fabl mutants, implying that other mechanisms of resistance are available (and that triclosan has targets other than Fabl). We have shown that triclosan induces the leakage of potassium ions from cells, an indication that triclosan targets the cytoplasmic membrane. However, whilst reduced susceptibility to triclosan did confer reduced susceptibility to the lethal effects of 7.5 mg/L triclosan, this effect was ameliorated by higher concentrations of triclosan. Indeed, in-use concentrations of the commercial preparation of triclosan, Irgacide LP 10, are equally active against reduced susceptibility <italic> S. aureus</italic> and wild-type. Therefore, the evolution of reduced-susceptibility to triclosan is of ambiguous clinical significance. We found that spontaneous mutation to reduced susceptibility was not associated with a significant fitness cost, augmenting its potential for emergence in nature. Evolution of reduced susceptibility did not confer co-resistance to other antimicrobials and MRSA and MSSA strains were equally susceptible. An assessment of commensal <italic> S. aureus</italic> carried amongst the student population of Cardiff revealed that reduced susceptibility to triclosan is rare in this population. However, coagulase-negative staphylococci (CoNS) showed consistently higher MICs for triclosan and may represent an amenable reservoir of resistance. There was no indication that mupirocin and triclosan resistance have co-transferred in the past. Indeed, there appeared to be no relationship between resistance to either of these compounds in <italic>S. aureus</italic>. Reduced susceptibility to triclosan could not be disseminated amongst 5". aureus, or related Gram-positive bacteria by transduction, conjugation or transformation. Importantly, during the course of this work we discovered that triclosan could select for <italic> S. aureus</italic> small-colony variants (SCVs) that were coincidently resistant to gentamicin and penicillin. These were slow growing and illustrated the typical SCV phenotype. SCVs were more readily transformable than wild-type cells and may represent an enduring reservoir of resistance determinants. In conclusion, we found that <italic>S. aureus</italic> could develop reduced susceptibility to triclosan by spontaneous mutation or the evolution of SCVs. However, the level of resistance is of ambiguous significance. We propose that triclosan does target Fabl, but also has other cellular targets, particularly at higher concentrations. Whilst the evolution of reduced susceptibility and the occurrence of SCVs should be monitored these should not preclude the use of triclosan as part of infection control procedures. However, to reduce the opportunities for resistance, infection control procedures should not rely upon a single antimicrobial to provide the panacea for nosocomial infections.