Multiple genotypes of Chlamydia pneumoniae identified in human carotid plaque

Chlamydia pneumoniae is an obligate intracellular respiratory pathogen that causes 10% of community-acquired pneumonia and has been associated with cardiovascular disease. Both whole-genome sequencing and specific gene typing suggest that there is relatively little genetic variation in human isolates of C. pneumoniae. To date, there has been little genomic analysis of strains from human cardiovascular sites. The genotypes of C. pneumoniae present in human atherosclerotic carotid plaque were analysed and several polymorphisms in the variable domain 4 (VD4) region of the outer-membrane protein-A (ompA) gene and the intergenic region between the ygeD and uridine kinase (ygeD-urk) genes were found. While one genotype was identified that was the same as one reported previously in humans (respiratory and cardiovascular), another genotype was found that was identical to a genotype from non-human sources (frog/koala).

Recommendations for animal DNA forensic and identity testing

Genetic analysis in animals has been used for many applications, such as kinship analysis, for determining the sire of an offspring when a female has been exposed to multiple males, determining parentage when an animal switches offspring with another dam, extended lineage reconstruction, estimating inbreeding, identification in breed registries, and speciation. It now also is being used increasingly to characterize animal materials in forensic cases. As such, it is important to operate under a set of minimum guidelines that assures that all service providers have a template to follow for quality practices. None have been delineated for animal genetic identity testing. Based on the model for human DNA forensic analyses, a basic discussion of the issues and guidelines is provided for animal testing to include analytical practices, data evaluation, nomenclature, allele designation, statistics, validation, proficiency testing, lineage markers, casework files, and reporting. These should provide a basis for professional societies and/or working groups to establish more formalized recommendations.

Methicillin-resistant Staphylococcus aureus genotyping using a small set of polymorphisms

The aim of this study was to identify a set of genetic polymorphisms that efficiently divides methicillin-resistant Staphylococcus aureus (MRSA) strains into groups consistent with the population structure. The rationale was that such polymorphisms could underpin rapid real-time PCR or low-density array-based methods for monitoring MRSA dissemination in a cost-effective manner. Previously, the authors devised a computerized method for identifying sets of single nucleoticle polymorphisms (SNPs) with high resolving power that are defined by multilocus sequence typing (MLST) databases, and also developed a real-time PCR method for interrogating a seven-member SNP set for genotyping S. aureus. Here, it is shown that these seven SNPs efficiently resolve the major MRSA lineages and define 27 genotypes. The SNP-based genotypes are consistent with the MRSA population structure as defined by eBURST analysis. The capacity of binary markers to improve resolution was tested using 107 diverse MRSA isolates of Australian origin that encompass nine SNP-based genotypes. The addition of the virulence-associated genes cna, pvl and bbplsdrE, and the integrated plasmids pT181, p1258 and pUB110, resolved the nine SNP-based genotypes into 21 combinatorial genotypes. Subtyping of the SCCmec locus revealed new SCCmec types and increased the number of combinatorial genotypes to 24. It was concluded that these polymorphisms provide a facile means of assigning MRSA isolates into well-recognized lineages.

Staphylococcus aureus genotyping using novel real-time PCR formats

One approach to microbial genotyping is to make use of sets of single-nucleotide polymorphisms (SNPs) in combination with binary markers. Here we report the modification and automation of a SNP-plus-binary-marker-based approach to the genotyping of Staphylococcus aureus and its application to 391 S. aureus isolates from southeast Queensland, Australia. The SNPs used were arcC210, tpi243, arcC162, gmk318, pta294, tpi36, tpi241, and pta383. These provide a Simpson's index of diversity (D) of 0.95 with respect to the S. aureus multilocus sequence typing database and define 61 genotypes and the major clonal complexes. The binary markers used were pvl, cna, sdrE, pT181, and pUB110. Two novel real-time PCR formats for interrogating these markers were compared. One of these makes use of light upon extension (LUX) primers and biplexed reactions, while the other is a streamlined modification of kinetic PCR using SYBR green. The latter format proved to be more robust. In addition, automated methods for DNA template preparation, reaction setup, and data analysis were developed. A single SNP-based method for ST-93 (Queensland clone) identification was also devised. The genotyping revealed the numerical importance of the South West Pacific and Queensland community-acquired methicillin-resistant S. aureus (MRSA) clones and the clonal complex 239 Aus-1/Aus-2 hospital-associated MRSA. There was a strong association between the community-acquired clones and pvl.

Methicillin-resistant Staphylococcus aureus in the Australian community: an evolving epidemic

Objective: To describe antimicrobial resistance and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolated in community settings in Australia. Design and setting: Survey of S. aureus isolates collected prospectively Australia-wide between July 2004 and February 2005; results were compared with those of similar surveys conducted in 2000 and 2002. Main outcome measures: Up to 100 consecutive, unique clinical isolates of S. aureus from outpatient settings were collected at each of 22 teaching hospital and five private laboratories from cities in all Australian states and territories. They were characterised by antimicrobial susceptibilities (by agar dilution methods), coagulase gene typing, pulsed-field gel electrophoresis, multilocus sequence typing, SCCmec typing and polymerase chain reaction tests for Panton-Valentine leukocidin (PVL) gene. Results: 2652 S. aureus isolates were collected, of which 395 (14.9%) were MRSA. The number of community-associated MRSA (CA-MRSA) isolates rose from 4.7% (118/2498) of S. aureus isolates in 2000 to 7.3% (194/2652) in 2004 (P=0.001). Of the three major CA-MRSA strains, WA-1 constituted 45/257 (18%) of MRSA in 2000 and 64/395 (16%) in 2004 (P=0.89), while the Queensland (OLD) strain increased from 13/257 (5%) to 58/395 (15%) (P=0.0004), and the south-west Pacific (SWP) strain decreased from 33/257 (13%) to 26/395 (7%) (P=0.01). PVL genes were detected in 90/195 (46%) of CA-MRSA strains, including 5/64 (8%) of WA-1, 56/58 (97%) of OLD, and 25/26 (96%) of SWP strains. Among health care-associated MRSA strains, all AUS-2 and AUS-3 isolates were multidrug-resistant, and UK EMRSA-15 isolates were resistant to ciprofloxacin and erythromycin (50%) or to ciprofloxacin alone (44%). Almost all (98%) of CA-MRSA strains were non-multiresistant. Conclusions: Community-onset MRSA continues to spread throughout Australia. The hypervirulence determinant PVL is often found in two of the most common CA-MRSA strains. The rapid changes in prevalence emphasise the importance of ongoing surveillance.

A type system with usage aspects

Linear typing schemes can be used to guarantee non-interference and so the soundness of in-place update with respect to a functional semantics. But linear schemes are restrictive in practice, and more restrictive than necessary to guarantee soundness of in-place update. This limitation has prompted research into static analysis and more sophisticated typing disciplines to determine when in-place update may be safely used, or to combine linear and non-linear schemes. Here we contribute to this direction by defining a new typing scheme that better approximates the semantic property of soundness of in-place update for a functional semantics. We begin from the observation that some data are used only in a read-only context, after which it may be safely re-used before being destroyed. Formalising the in-place update interpretation in a machine model semantics allows us to refine this observation, motivating three usage aspects apparent from the semantics that are used to annotate function argument types. The aspects are (1) used destructively, (2), used read-only but shared with result, and (3) used read-only and not shared with the result. The main novelty is aspect (2), which allows a linear value to be safely read and even aliased with a result of a function without being consumed. This novelty makes our type system more expressive than previous systems for functional languages in the literature. The system remains simple and intuitive, but it enjoys a strong soundness property whose proof is non-trivial. Moreover, our analysis features principal types and feasible type reconstruction, as shown in M. Konen'y (In TYPES 2002 workshop, Nijmegen, Proceedings, Springer-Verlag, 2003).

An outbreak of Serratia marcescens on the neonatal unit:a tale of two clones

Serratia spp. are an important cause of hospital-acquired infections and outbreaks in high-risk settings. Twenty-one patients were infected or colonized over a nine-month period during 2001-2002 on a neonatal unit. Twenty-two isolates collected were examined for antibiotic susceptibility, β-lactamase production and genotype. Random-amplified polymorphic DNA polymerase chain reaction and pulsed-field gel electrophoresis revealed that two clones were present. The first clone caused invasive clinical infection in four babies, and was subsequently replaced by a non-invasive clone that affected 14 babies. Phenotypically, the two strains also differed in their prodigiosin production; the first strain was non-pigmented whereas the second strain displayed pink-red pigmentation. Clinical features suggested a difference in their pathogenicity. No environmental source was found. The outbreak terminated following enhanced compliance with infection control measures and a change of antibiotic policy. Although S. marcescens continued to be isolated occasionally for another five months of follow-up, these were sporadic isolates with distinct molecular typing patterns. © 2005 The Hospital Infection Society.

Analysis of clinical isolates of Propionibacterium acnes by optimised RAPD

Random amplification of polymorphic DNA (RAPD) was evaluated as a genotypic method for typing clinical strains of Propionibacterium acnes. RAPD can suffer from problems of reproducibility if parameters are not standardised. In this study the reaction conditions were optimised by adjusting template DNA concentration and buffer constituents. All isolates were typeable using the optimised RAPD protocol which was found to be highly discriminatory (Simpson's diversity index, 0.98) and reproducible. Typing of P. acnes by optimised RAPD is an invaluable tool for the epidemiological investigation of P. acnes for which no other widely accepted method currently exists. © 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Spatial aspects of MRSA epidemiology:A case study using stochastic simulation, kernel estimation and SaTScan

The identification of disease clusters in space or space-time is of vital importance for public health policy and action. In the case of methicillin-resistant Staphylococcus aureus (MRSA), it is particularly important to distinguish between community and health care-associated infections, and to identify reservoirs of infection. 832 cases of MRSA in the West Midlands (UK) were tested for clustering and evidence of community transmission, after being geo-located to the centroids of UK unit postcodes (postal areas roughly equivalent to Zip+4 zip code areas). An age-stratified analysis was also carried out at the coarser spatial resolution of UK Census Output Areas. Stochastic simulation and kernel density estimation were combined to identify significant local clusters of MRSA (p<0.025), which were supported by SaTScan spatial and spatio-temporal scan. In order to investigate local sampling effort, a spatial 'random labelling' approach was used, with MRSA as cases and MSSA (methicillin-sensitive S. aureus) as controls. Heavy sampling in general was a response to MRSA outbreaks, which in turn appeared to be associated with medical care environments. The significance of clusters identified by kernel estimation was independently supported by information on the locations and client groups of nursing homes, and by preliminary molecular typing of isolates. In the absence of occupational/ lifestyle data on patients, the assumption was made that an individual's location and consequent risk is adequately represented by their residential postcode. The problems of this assumption are discussed, with recommendations for future data collection.

Characterisation of Aeromonas strains and species by pulsed field gel electrophoresis and principal components analysis

Aeromonas genomes were investigated by restriction digesting chromosomal DNA with the endonuclease XbaI, separation of restriction fragments by pulsed field gel electrophoresis (PFGE) and principal components analysis (PCA) of resulting separation patterns. A. salmonicida salmonicida were unique amongst the isolates investigated. Separation profiles of these isolates were similar and all characterised by a distinct absence of bands in the 250kb region. Principal components analysis represented these strains as a clearly defined homogeneous group separated by insignificant Euclidian distances. However, A. salmonicida achromogenes isolates in common with those of A. hydrophila and A. sobria were shown by principal components analysis to be more heterogeneous in nature. Fragments from these isolates were more uniform in size distribution but as demonstrated by the Euclidian distances attained through PCA potentially characteristic of each strain. Furthermore passaging of Aeromonas isolates through an appropriate host did not greatly modify fragment separation profiles, indicative of the genomic stability of test aeromonads and the potential of restriction digesting/PFGE/PCA in Aeromonas typing.

Phenotypic and genotypic characterisation of Clostrum Difficile

Clostridium difficile is at present one of the most common nosocomial infections in the developed world. Hypervirulent strains (PCR ribotype 027) of C. difficile which produce enhanced levels of toxins have also been associated with other characteristics such as a greater rate of sporulation and resistance to fluoroquinolones. Infection due to C. difficile PCR ribotype 027 has also been associated with greater rates of morbidity and mortality. The aim of this thesis was to investigate both the phenotypic and genotypic characteristics of two populations of toxigenic clinical isolates of C. difficile which were recovered from two separate hospital trusts within the UK. Phenotypic characterisation of the isolates was undertaken using analytical profile indexes (APIs), minimum inhibitory concentrations(MICs) and S-layer protein typing. In addition to this, isolates were also investigated for the production of a range of extracellular enzymes as potential virulence factors. Genotypic characterisation was performed using a random amplification of polymorphic DNA(RAPD) PCR protocol which was fully optimised in this study, and the gold standard method, PCR ribotyping. The discriminatory power of both methods was compared and the similarity between the different isolates also analysed. Associations between the phenotypic and genotypic characteristics and the recovery location of the isolate were then investigated. Extracellular enzyme production and API testing revealed little variation between the isolates; with S-layer typing demonstrating low discrimination. Minimum inhibitory concentrations did not identify any resistance towards either vancomycin or metronidazole; there were however significant differences in the distribution of antibiogram profiles of isolates recovered from the two different trusts. The RAPD PCR protocol was successfully optimised and alongside PCR ribotyping, effectively typed all of the clinical isolates and also identified differences in the number of types defined between the two locations. Both PCR ribotyping and RAPD demonstrated similar discriminatory power; however, the two genotyping methods did not generate amplicons that mapped directly onto each other and therefore clearly characterised isolates based on different genomic markers. The RAPD protocol also identified different subtypes within PCR ribotypes, therefore demonstrating that all isolates defined as a particular PCR ribotype were not the same strain. No associations could be demonstrated between the phenotypic and genotypic characteristics observed; however, the location from which an isolate was recovered did appear to influence antibiotic resistance and genotypic characteristics. The phenotypic and genotypic characteristics observed amongst the C. difficile isolates in this study, may provide a basis for the identification of further targets which may be potentially incorporated into future methods for the characterisation of C. difficile isolates.

The prevention and diagnosis of central venous catheter-related infection

The potential source of CVC colonisation was assessed. Isolates of coagulase-negative staphylococci (CoNS) recovered from the skin and CVC components of 3 cardiothoracic surgery patients were characterised by pulsed-field gel electrophoresis (PFGE). The genetic heterogeneity of CoNS isolated from the skin was demonstrated and specific genotypes implicated in catheter colonisation. In addition, phenotypic and genotypic typing techniques were assessed for their ability to characterise strains of CoNS recovered from 33 patients who developed catheter-related bloodstream infection (CR-BSI) on a bone marrow transplant (BMT) unit and Siaphylococcus aureus recovered from 6 cardiothoracic surgery patients with surgical site infection (SSI) following median sternotomy. This epidemiological investigation revealed that common strains of CoNS and 51 aureus where not associated with infection in patients with CR-BSI or sternal SSI during the study period. Furthermore, there was no correlation between phenotypic and genotypic characterisation results. The variable expression of phenotypic traits within strains of staphylococci was evident whilst PFGE and randomly amplified polymorphic DNA (RAPD) were highly discriminatory for the molecular characterisation of S. aureus and CoNS. This was highlighted in 8 stem cell transplant (SCT) patients whereby it was demonstrated that routine identification and characterisation of CoNS by phenotypic techniques may not be adequate for the diagnosis of CR-BSI by current guidelines. The potential of the lipid S ELISA to facilitate the diagnosis of CR-BSI in 38 haematology/SCT patients and sternal SSI in 57 cardiothoracic surgery patients was also assessed. The ELISA proved to be a sensitive test for the rapid serodiagnosis of infection due to staphylococci in immunocompetent patients. The acridine orange leucocyte cytospin test (AOLC) was also evaluated for the rapid diagnosis of CR-BSI in 16 haematology/SCT patients with Hickman CVC in situ. Although the sensitivity of the test was low, it may provide a useful adjunct to conventional methods for the in situ sampling of catheters to predict and diagnose CR-BSI, preventing the unnecessary removal of CVC.

Characterisation of propionibacterium acnes

Propionibacterium acnes forms part of the normal flora of the skin, oral cavity, large intestine and the external ear. Historically, P. acnes is considered to be of low virulence; however, in recent years it has been found as the aetiological agent in various pathologies including acne vulgaris, endophthalmitis, endocarditis, osteomyelitis, sarcoidosis, prosthetic hip infections and sciatica. It currently remains unclear why this normally harmless commensal can cause infection and contribute to a number of clinically significant conditions. This thesis has sought to investigate the phenotypic, genetic and antigenic properties of P.acnes strains isolated from sciatica patients undergoing microdiscectomy, normal skin, blood cultures, prosthetic hips and acne lesions. Isolates' phenotype was examined by determining their biotype by analytical profile index, antimicrobial susceptibility, virulence factor expression and serotype. A molecular typing method for P.acnes was developed using random amplification of polymorphic DNA (RAPD). Patient serum was used to screen P.acnes strains for antigens expressed in vivo and the chemical composition determined. The serodiagnostic potential and inflammatory properties of identified antigens were assessed. The optimised and reproducible RAPD protocol classified strains into three major clusters and was found to distinguish between the serotypes I and II for a large number of clinical isolates. Molecular typing by RAPD also enabled the identification of a genotype that did not react with the type I or II monoclonal antibodies and these strains may therefore constitute a previously undiscovered subspecies of P.acnes with a genetic background different from the type I and II serotypes. A major cell associated antigen produced by all strains was identified and characterised. A serological assay based on the antigen was used to measure IgG and IgM levels in serum from patients with acne, sciatica and controls. No difference in levels of antibodies was detected. Inflammatory properties of the antigen were measured by exposing murine macrophage-like cells and measuring the release of nitric oxide and tumour necrosis factor-alpha (TNF-α). Only TNF-α was elicited in response to the antigen. The phenotypic, genotypic and antigenic properties of this organism may provide a basis for future studies on P.acnes virulence and provide an insight into its mechanisms of pathogenesis.

Salmonella in companion animals

In an increasingly hygiene concerned society, a major barrier to pet ownership is the perceived role of companion animals in contributing to the risk of exposure to zoonotic bacterial pathogens, such as Salmonella. Manifestations of Salmonella can range from acute gastroenteritis to perfuse enteric fever, in both humans and dogs. Dogs are heavily associated with asymptomatic carriage of Salmonella as the microorganism can persist in the lower intestines of this host which can be then excreted into the environment. Studies in to the asymptomatic carriage of Salmonella in dogs are somewhat dated and there is limited UK data. The current UK carriage rate in dogs was investigated in a randomised dog population and it was revealed that the carriage rate in this population was very low with only one household dog positive for the carriage of Salmonella enterica arizonae (0.2%), out of 490 dogs sampled. Salmonella serotypes share phenotypic and genotypic similarities which are captured in epidemiological typing methods. Therefore, in parallel to the epidemiological investigations, a panel of clinical canine (VLA, UK) and human (Aston University, UK) Salmonella isolates were profiled based on their phenotypic and genotypic characteristics; using API 20E, Biolog Microbial ID System, antibiotic sensitivity testing and PFGE, respectively. Antibiotic sensitivity testing revealed a significant difference between the canine and human isolates with the canine group demonstrating a higher resistance to the panel of antibiotics tested. Further metabolic capabilities of the strains were tested using the Biolog Microbial ID System, which reveal no clear association between the two host groups. However, coupled with Principle Component Analysis two canine isolates were discriminated from the entire population on the basis of a high up-regulation of two carbohydrates. API 20E testing revealed no association between the two host groups. A PFGE harmonised protocol was used to genotypically profile the strains. A dendrogram depicting PFGE profiles of the panel of Salmonella isolates was performed where similarities were calculated by Dice coefficient and represented by UPGMA clustering. Clustering of the profiles from canine isolates and human isolates (HPA, UK) was diverse representing a natural heterogeneity of the genus, additionally, no clear clustering of the isolates was observed between host groups. Clustering was observed with isolates from the same serotype, independent of host origin. Host adaption is a common phenomenon in certain Salmonella serotypes, for example S. Typhi in humans and S. Dublin in cattle. It was of interest to investigate potential host adaptive or restricted strains for canine host by performing adhesion and invasion assays on Dog Intestinal Epithelial Cells (DIECs) (WALTHAM®, UK) and human CaCo-2 (HPA, UK) cell lines. Salmonella arizonae and Enteritidis from an asymptomatic dog and clinical isolate, respectively, demonstrated a significantly high proportion of invasion in DIEC in comparison to human CaCo-2 cells and other tested Salmonella serotypes. This may be suggestive of a potential host restrictive strain as their ability to invade the CaCo-2 cell line was significantly lower than the other serotypes. In conclusion to this thesis the investigations carried out suggest that asymptomatic carriage of Salmonella in UK dogs is low however the microorganism remains as a zoonotic and anthroponotic pathogen based on phenotypic and genotypic characterisation however there may be potential for particular serotype to become host restricted as observed in invasion assays