High-resolution genotyping of Lymphogranuloma Venereum (LGV) strains of Chlamydia trachomatis in London using multi-locus VNTR analysis-ompA genotyping (MLVA-ompA).

BACKGROUND: Lymphogranuloma venereum (LGV) is caused by Chlamydia trachomatis strains with ompA genotypes L1 to L3. An LGV epidemic associated with the L2b genotype has emerged in the past few decades amongst men who have sex with men (MSM). C. trachomatis genotypes can be discriminated by outer membrane protein A gene (ompA) sequencing, however this method has limited resolution. This study employed a high-resolution genotyping method, namely, multi-locus tandem repeat (VNTR) analysis with ompA sequencing (MLVA-ompA), to assess the distribution of LGV MLVA-ompA genotypes amongst individuals attending genitourinary medicine (GUM) clinics in London. METHODS: Clinical specimens were collected from individuals attending eight London-based GUM clinics. Specimens that tested positive for C. trachomatis by commercial nucleic acid amplification test (NAAT) were confirmed as LGV by pmpH real-time PCR. LGV-positive DNA extracts were subsequently genotyped using MLVA-ompA. RESULTS: Two hundred and thirty DNA extracts were confirmed as LGV, and 162 (70%) yielded complete MLVA-ompA genotypes. Six LGV MLVA-ompA genotypes were identified: 1.9.2b-L2, 1.9.3b-L2b, 1.9.2b-L2b, 1.9.2b-L2b/D, 1.4a.2b-L2b, and 5.9.2b-L1. The following LGV ompA genotypes were identified (in descending order of abundance): L2, L2b, L2b/D, and L1. Eight ompA sequences with the hybrid L2b/D profile were detected. The hybrid sequence was identical to the ompA of a recombinant L2b/D strain detected in Portugal in 2017. CONCLUSIONS: The L2 ompA genotype was found to predominate in the London study population. The study detected an unusual hybrid L2b/D ompA profile that was previously reported in Portugal. We recommend further monitoring and surveillance of LGV strains within the UK population.

The Nature and Extent of Plasmid Variation in Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular pathogen of humans, causing both the sexually transmitted infection, chlamydia, and the most common cause of infectious blindness, trachoma. The majority of sequenced C. trachomatis clinical isolates carry a 7.5-Kb plasmid, and it is becoming increasingly evident that this is a key determinant of pathogenicity. The discovery of the Swedish New Variant and the more recent Finnish variant highlight the importance of understanding the natural extent of variation in the plasmid. In this study we analysed 524 plasmid sequences from publicly available whole-genome sequence data. Single nucleotide polymorphisms (SNP) in each of the eight coding sequences (CDS) were identified and analysed. There were 224 base positions out of a total 7550 bp that carried a SNP, which equates to a SNP rate of 2.97%, nearly three times what was previously calculated. After normalising for CDS size, CDS8 had the highest SNP rate at 3.97% (i.e., number of SNPs per total number of nucleotides), whilst CDS6 had the lowest at 1.94%. CDS5 had the highest total number of SNPs across the 524 sequences analysed (2267 SNPs), whereas CDS6 had the least SNPs with only 85 SNPs. Calculation of the genetic distances identified CDS6 as the least variable gene at the nucleotide level (d = 0.001), and CDS5 as the most variable (d = 0.007); however, at the amino acid level CDS2 was the least variable (d = 0.001), whilst CDS5 remained the most variable (d = 0.013). This study describes the largest in-depth analysis of the C. trachomatis plasmid to date, through the analysis of plasmid sequence data mined from whole genome sequences spanning 50 years and from a worldwide distribution, providing insights into the nature and extent of existing variation within the plasmid as well as guidance for the design of future diagnostic assays. This is crucial at a time when single-target diagnostic assays are failing to detect natural mutants, putting those infected at risk of a serious long-term and life-changing illness.

Development and Assessment of a Diagnostic DNA Oligonucleotide Microarray for Detection and Typing of Meningitis-Associated Bacterial Species.

Meningitis is commonly caused by infection with a variety of bacterial or viral pathogens. Acute bacterial meningitis (ABM) can cause severe disease, which can progress rapidly to a critical life-threatening condition. Rapid diagnosis of ABM is critical, as this is most commonly associated with severe sequelae with associated high mortality and morbidity rates compared to viral meningitis, which is less severe and self-limiting. We have designed a microarray for detection and diagnosis of ABM. This has been validated using randomly amplified DNA targets (RADT), comparing buffers with or without formamide, in glass slide format or on the Alere ArrayTubeTM (Alere Technologies GmbH) microarray platform. Pathogen-specific signals were observed using purified bacterial nucleic acids and to a lesser extent using patient cerebral spinal fluid (CSF) samples, with some technical issues observed using RADT and glass slides. Repurposing the array onto the Alere ArrayTubeTM platform and using a targeted amplification system increased specific and reduced nonspecific hybridization signals using both pathogen nucleic and patient CSF DNA targets, better revealing pathogen-specific signals although sensitivity was still reduced in the latter. This diagnostic microarray is useful as a laboratory diagnostic tool for species and strain designation for ABM, rather than for primary diagnosis.

The Chlamydia muridarum plasmid revisited : new insights into growth kinetics.

Background: Research in chlamydial genetics is challenging because of its obligate intracellular developmental cycle. In vivo systems exist that allow studies of different aspects of basic biology of chlamydiae, the murine Chlamydia muridarum model is one of great importance and thus an essential research tool. C. muridarum carries a plasmid that has a role in virulence.  Our aim was to compare and contrast the C. muridarum plasmid-free phenotype with that of a chromosomally isogenic plasmid-bearing strain, through the inclusion phase of the developmental cycle. Methods: We measured infectivity for plasmid bearing and plasmid-cured C. muridarum by inclusion forming assays in McCoy cells and in parallel bacterial chromosome replication by quantitative PCR, throughout the developmental cycle. In addition to these studies, we have carefully monitored chlamydial inclusion formation by confocal microscopy and transmission electron microscopy. A new E.coli/chlamydial shuttle vector (pNigg::GFP) was constructed using standard cloning technology and used to transform C. muridarum for further phenotypic studies. Results: We have advanced the definition of the chlamydial phenotype away from the simple static observation of mature inclusions and redefined the C. muridarum plasmid-based phenotype on growth profile and inclusion morphology. Our observations on the growth properties of plasmid-cured C. muridarum challenge the established interpretations, especially with regard to inclusion growth kinetics. Introduction of the shuttle plasmid pNigg::GFP into plasmid-cured C. muridarum restored the wild-type plasmid-bearing phenotype and confirmed that loss of the plasmid was the sole cause for the changes in growth and chromosomal replication. Conclusions: Accurate growth curves and sampling at multiple time points throughout the developmental cycle is necessary to define plasmid phenotypes.  There are subtle but important (previously unnoticed) differences in the overall growth profile of plasmid-bearing and plasmid-free C. muridarum.  We have proven that the differences described are solely due to the plasmid pNigg.

Detailed molecular epidemiology of Chlamydia trachomatis in the population of Southampton attending the genitourinary medicine clinic in 2012-13 reveals the presence of long established genotypes and transitory sexual networks.

Chlamydia trachomatis is the most common sexually transmitted infection (STI) in England. Our objective was to perform a detailed survey of the molecular epidemiology of C. trachomatis in the population of Southampton UK attending the genitourinary medicine clinic (GUM) to seek evidence of sexual network activity. Our hypothesis was that certain genotypes can be associated with specific demographic determinants. 380 positive samples were collected from 375 C. trachomatis positive GUM attendees out of the 3118 who consented to be part of the survey. 302 of the positive samples were fully genotyped. All six of the predominant genotypes possessed ompA locus type E. One ward of Southampton known to contain a large proportion of students had a different profile of genotypes compared to other areas of the city. Some genotypes appeared embedded in the city population whilst others appeared transient. Predominant circulating genotypes remain stable within a city population whereas others are sporadic. Sexual networks could be inferred but not conclusively identified using the data from this survey.

Duration of intravenous antibiotic therapy for children with acute osteomyelitis or septic arthritis: a feasibility study.

BACKGROUND: There is little current consensus regarding the route or duration of antibiotic treatment for acute osteomyelitis (OM) and septic arthritis (SA) in children. OBJECTIVE: To assess the overall feasibility and inform the design of a future randomised controlled trial (RCT) to reduce the duration of intravenous (i.v.) antibiotic use in paediatric OM and SA. DESIGN: (1) A prospective service evaluation (cohort study) to determine the current disease spectrum and UK clinical practice in paediatric OM/SA; (2) a prospective cohort substudy to assess the use of targeted polymerase chain reaction (PCR) in diagnosing paediatric OM/SA; (3) a qualitative study to explore families' views and experiences of OM/SA; and (4) the development of a core outcome set via a systematic review of literature, Delphi clinician survey and stakeholder consensus meeting. SETTING: Forty-four UK secondary and tertiary UK centres (service evaluation). PARTICIPANTS: Children with OM/SA. INTERVENTIONS: PCR diagnostics were compared with culture as standard of care. Semistructured interviews were used in the qualitative study. RESULTS: Data were obtained on 313 cases of OM/SA, of which 218 (61.2%) were defined as simple disease and 95 (26.7%) were defined as complex disease. The epidemiology of paediatric OM/SA in this study was consistent with existing European data. Children who met oral switch criteria less than 7 days from starting i.v. antibiotics were less likely to experience treatment failure (9.6%) than children who met oral switch criteria after 7 days of i.v. therapy (16.1% when switch was between 1 and 2 weeks; 18.2% when switch was > 2 weeks). In 24 out of 32 simple cases (75%) and 8 out of 12 complex cases (67%) in which the targeted PCR was used, a pathogen was detected. The qualitative study demonstrated the importance to parents and children of consideration of short- and long-term outcomes meaningful to families themselves. The consensus meeting agreed on the following outcomes: rehospitalisation or recurrence of symptoms while on oral antibiotics, recurrence of infection, disability at follow-up, symptom free at 1 year, limb shortening or deformity, chronic OM or arthritis, amputation or fasciotomy, death, need for paediatric intensive care, and line infection. Oral switch criteria were identified, including resolution of fever for ≥ 48 hours, tolerating oral food and medicines, and pain improvement. LIMITATIONS: Data were collected in a 6-month period, which might not have been representative, and follow-up data for long-term complications are limited. CONCLUSIONS: A future RCT would need to recruit from all tertiary and most secondary UK hospitals. Clinicians have implemented early oral switch for selected patients with simple disease without formal clinical trial evidence of safety. However, the current criteria by which decisions to make the oral switch are made are not clearly established or evidence based. FUTURE WORK: A RCT in simple OM and SA comparing shorter- or longer-course i.v. therapy is feasible in children randomised after oral switch criteria are met after 7 days of i.v. therapy, excluding children meeting oral switch criteria in the first week of i.v. therapy. This study design meets clinician preferences and addresses parental concerns not to randomise prior to oral switch criteria being met. FUNDING: The National Institute for Health Research Health Technology Assessment programme.

Comprehensive global genome dynamics of Chlamydia trachomatis show ancient diversification followed by contemporary mixing and recent lineage expansion.

Chlamydia trachomatis is the world's most prevalent bacterial sexually transmitted infection and leading infectious cause of blindness, yet it is one of the least understood human pathogens, in part due to the difficulties of in vitro culturing and the lack of available tools for genetic manipulation. Genome sequencing has reinvigorated this field, shedding light on the contemporary history of this pathogen. Here, we analyze 563 full genomes, 455 of which are novel, to show that the history of the species comprises two phases, and conclude that the currently circulating lineages are the result of evolution in different genomic ecotypes. Temporal analysis indicates these lineages have recently expanded in the space of thousands of years, rather than the millions of years as previously thought, a finding that dramatically changes our understanding of this pathogen's history. Finally, at a time when almost every pathogen is becoming increasingly resistant to antimicrobials, we show that there is no evidence of circulating genomic resistance in C. trachomatis.

Development and evaluation of an enzyme-linked immunosorbent assay for the detection of antibodies to a common urogenital derivative of Chlamydia trachomatis plasmid-encoded PGP3.

BACKGROUND: Urogenital infection with Chlamydia trachomatis is the most commonly diagnosed sexually transmitted infection in the developed world. Accurate measurement and therefore understanding the seroprevalence of urogenital C. trachomatis infections requires a rigorously optimised and validated ELISA. Previous ELISAs based on the C. trachomatis plasmid-encoded protein, PGP3, have been described but lack standardisation and critical controls or use a less common PGP3 as the capture antigen. METHODOLOGY/PRINCIPAL FINDINGS: A sensitive and specific indirect ELISA was developed based on recombinant PGP3 derived from a urogenital strain of C. trachomatis, serovar E (pSW2), using a rigorous validation protocol. Serum samples were collected from 166 genitourinary medicine (GUM) clinic patients diagnosed as positive or negative for urogenital C. trachomatis infection by nucleic acid amplification testing (NAATs). Overall sensitivity and specificity compared to NAATs was 68.18% and 98.0%, respectively. Sensitivities for female and male samples were 71.93% and 64.15%, respectively. Comparison of samples from these patients diagnosed positive for C. trachomatis by NAAT and patients diagnosed negative by NAAT revealed statistical significance (p≤0.0001). CONCLUSIONS: We have developed and validated a sensitive and specific ELISA to detect anti-PGP3 antibodies as an indicator of past and current infection to C. trachomatis using PGP3 from a common urogenital strain. It is anticipated that this assay will be used for seroepidemiological analysis of urogenital C. trachomatis in populations.

Highly diverse MLVA-ompA genotypes of rectal Chlamydia trachomatis among men who have sex with men in Brighton, UK and evidence for an HIV-related sexual network.

OBJECTIVES: In this prospective study, we aimed to determine the distribution of genotypes by multilocus variable number tandem repeat (VNTR) analysis plus analysis of the ompA gene (MLVA-ompA) of rectal Chlamydia trachomatis among men who have sex with men (MSM) attending Brighton Genitourinary Medicine (GUM) Clinic and to examine any correlations with clinical variables, including HIV status, and to isolate rectal C. trachomatis cultures maximising the possibility of obtaining complete genotyping data. METHODS: Samples were assigned genotypes by PCR and sequencing of the markers of the MLVA-ompA genotyping system. Rectal C. trachomatis was isolated in cell culture using McCoy cells. Data regarding demographics, HIV status, rectal symptoms and history of sexually transmitted infections, including C. trachomatis, were collected. RESULTS: 1809 MSM attending the clinic between October 2011 and January 2013 took part in the study, 112 (6.2%) of whom had rectal samples that tested positive for C. trachomatis. 85/112 (75.9%) C. trachomatis-positive rectal samples were assigned 66 different genotypes. Two distinct genotype subclusters were identified: subcluster 1 consisted of more HIV-negative men than subcluster 2 (p=0.025), and the MLVA-ompA genotypes in these subclusters reflected this. Isolates were successfully cultured from 37 of the 112 specimens, from which 27 otherwise unobtainable (from direct PCR) MLVA-ompA genotypes were gained. CONCLUSIONS: The most prevalent genotypes were G, E and D representing some overlap with the heterosexual distribution in UK. Subcluster 1 consisted of more 'heterosexual genotypes' and significantly more HIV-negative men than subcluster 2, associated with 'MSM genotypes'. There was a higher diversity of C. trachomatis strains among MSM in Brighton than observed in other cities.

Rapid detection of diagnostic targets using isothermal amplification and HyBeacon probes--a homogenous system for sequence-specific detection.

Isothermal amplification is a rapid, simple alternative to PCR, with amplification commonly detected using fluorescently labelled oligonucleotide probes, intercalating dyes or increased turbidity as a result of magnesium pyrophosphate generation. SNP identification is possible but requires either allele-specific primers or multiple dye-labelled probes, but further downstream processing is often required for allelic identification. Here we demonstrate that modification of common isothermal amplification methods by the addition of HyBeacon probes permits homogeneous sequence detection and discrimination by melting or annealing curve analysis. Furthermore, we demonstrate that isothermal amplification and sequence discrimination is possible directly from a crude sample such as an expressed buccal swab.

Cryptic resistance in Staphylococcus aureus: a risk for the treatment of skin infection?

PURPOSE OF REVIEW: The purpose of this review is to explore major challenges that face routine diagnostic laboratories in detecting cryptic (hidden) antibiotic resistances in Staphylococcus aureus and the impact of these covert resistances in the management of skin and soft tissue infections. RECENT FINDINGS: The review covers recent literature and works regarding a number of evolving mechanisms and forms of antibiotic resistances in S. aureus, including novel oxacillin-susceptible-mecA-positive meticillin-resistant S. aureus (OS-MRSA) strains and MRSA isolates that harbour a divergent mecA homologue termed mecC within the novel staphylococcal cassette chromosome mec XI element. SUMMARY: S. aureus strains causing skin and soft tissue infections are evolving with regard to virulence as well as antimicrobial resistance. Cryptic resistances continue to escape routine diagnostic tests. This means that there are still many unknowns regarding their global dissemination, virulence, threats in clinical practice and optimal treatment strategies. Larger studies are needed to further understand the pathogenic consequences of cryptic resistances in S. aureus in skin and soft tissue infections, which may ultimately provide novel preventive or treatment approaches for this significant human pathogen.

Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA.

The study of bacterial populations using whole-genome sequencing is of considerable scientific and clinical interest. However, obtaining bacterial genomic information is not always trivial: the target bacteria may be difficult to culture or uncultured, and they may be found within samples containing complex mixtures of other contaminating microbes and/or host cells, from which it is very difficult to derive robust sequencing data. Here we describe our procedure to generate sufficient DNA for whole-genome sequencing from clinical samples and without the need for culture, as successfully used on the difficult-to-culture, obligate intracellular pathogen Chlamydia trachomatis. Our protocol combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification (WGA), which is followed by high-throughput sequencing. Compared with other techniques that might be used to generate such data, IMS-MDA is an inexpensive, low-technology and highly transferable process that provides amplified genomic DNA for sequencing from target bacteria in under 5 h, with little hands-on time.

Ascitic microbiota composition is correlated with clinical severity in cirrhosis with portal hypertension.

Identification of pathogenic bacteria in ascites correlates with poor clinical outcomes. Ascites samples are commonly reported culture-negative, even where frank infection is indicated. Culture-independent methods have previously reported bacterial DNA in ascites, however, whether this represents viable bacterial populations has not been determined. We report the first application of 16S rRNA gene pyrosequencing and quantitative PCR in conjunction with propidium monoazide sample treatment to characterise the viable bacterial composition of ascites. Twenty five cirrhotic patients undergoing paracentesis provided ascites. Samples were treated with propidium monoazide to exclude non-viable bacterial DNA. Total bacterial load was quantified by 16S rRNA Q-PCR with species identity and relative abundance determined by 16S rRNA gene pyrosequencing. Correlation of molecular microbiology data with clinical measures and diagnostic microbiology was performed. Viable bacterial signal was obtained in 84% of ascites samples, both by Q-PCR and pyrosequencing. Approximately 190,000 ribosomal pyrosequences were obtained, representing 236 species, including both gut and non gut-associated species. Substantial variation in the species detected was observed between patients. Statistically significant relationships were identified between the bacterial community similarity and clinical measures, including ascitic polymorphonuclear leukocyte count and Child-Pugh class. Viable bacteria are present in the ascites of a majority of patients with cirrhosis including those with no clinical signs of infection. Microbiota composition significantly correlates with clinical measures. Entry of bacteria into ascites is unlikely to be limited to translocation from the gut, raising fundamental questions about the processes that underlie the development of spontaneous bacterial peritonitis.

Staphylococcus aureus small-colony variants are independently associated with worse lung disease in children with cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) lung disease is associated with diverse bacteria chronically infecting the airways. Slow-growing, antibiotic-resistant mutants of Staphylococcus aureus known as small-colony variants (SCVs) have been isolated from respiratory secretions from European adults and children with CF lung disease using specific but infrequently used culture techniques. Staphylococcus aureus SCVs can be selected either by exposure to specific antibiotics or by growth with another CF pathogen, Pseudomonas aeruginosa. We sought to determine the prevalence, clinical significance, and likely mechanisms of selection of S. aureus SCVs among a US cohort of children with CF. METHODS: We performed a 2-year study of 100 children with CF using culture techniques sensitive for S. aureus SCVs, and evaluated associations with clinical characteristics using multivariable regression models. RESULTS: Staphylococcus aureus SCV infection was detected among 24% of participants and was significantly associated with a greater drop in lung function during the study (P = .007, adjusted for age and lung function at enrollment). This association persisted after adjusting for infection with other known CF pathogens, including P. aeruginosa and methicillin-resistant S. aureus. Evidence indicated that S. aureus SCVs were likely selected in vivo by treatment with the antibiotic trimethoprim-sulfamethoxazole and possibly by coinfection with P. aeruginosa. CONCLUSIONS: Infection with SCV S. aureus was independently associated with worse CF respiratory outcomes in this pediatric cohort. As many clinical microbiology laboratories do not specifically detect S. aureus SCVs, validation and extension of these findings would require widespread changes in the usual laboratory and clinical approaches to these bacteria.

Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture.

The use of whole-genome sequencing as a tool for the study of infectious bacteria is of growing clinical interest. Chlamydia trachomatis is responsible for sexually transmitted infections and the blinding disease trachoma, which affect hundreds of millions of people worldwide. Recombination is widespread within the genome of C. trachomatis, thus whole-genome sequencing is necessary to understand the evolution, diversity, and epidemiology of this pathogen. Culture of C. trachomatis has, until now, been a prerequisite to obtain DNA for whole-genome sequencing; however, as C. trachomatis is an obligate intracellular pathogen, this procedure is technically demanding and time consuming. Discarded clinical samples represent a large resource for sequencing the genomes of pathogens, yet clinical swabs frequently contain very low levels of C. trachomatis DNA and large amounts of contaminating microbial and human DNA. To determine whether it is possible to obtain whole-genome sequences from bacteria without the need for culture, we have devised an approach that combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification. Using IMS-MDA in conjunction with high-throughput multiplexed Illumina sequencing, we have produced the first whole bacterial genome sequences direct from clinical samples. We also show that this method can be used to generate genome data from nonviable archived samples. This method will prove a useful tool in answering questions relating to the biology of many difficult-to-culture or fastidious bacteria of clinical concern.

Discovery of a novel class of boron-based antibacterials with activity against gram-negative bacteria.

Gram-negative bacteria cause approximately 70% of the infections in intensive care units. A growing number of bacterial isolates responsible for these infections are resistant to currently available antibiotics and to many in development. Most agents under development are modifications of existing drug classes, which only partially overcome existing resistance mechanisms. Therefore, new classes of Gram-negative antibacterials with truly novel modes of action are needed to circumvent these existing resistance mechanisms. We have previously identified a new a way to inhibit an aminoacyl-tRNA synthetase, leucyl-tRNA synthetase (LeuRS), in fungi via the oxaborole tRNA trapping (OBORT) mechanism. Herein, we show how we have modified the OBORT mechanism using a structure-guided approach to develop a new boron-based antibiotic class, the aminomethylbenzoxaboroles, which inhibit bacterial leucyl-tRNA synthetase and have activity against Gram-negative bacteria by largely evading the main efflux mechanisms in Escherichia coli and Pseudomonas aeruginosa. The lead analogue, AN3365, is active against Gram-negative bacteria, including Enterobacteriaceae bearing NDM-1 and KPC carbapenemases, as well as P. aeruginosa. This novel boron-based antibacterial, AN3365, has good mouse pharmacokinetics and was efficacious against E. coli and P. aeruginosa in murine thigh infection models, which suggest that this novel class of antibacterials has the potential to address this unmet medical need.

Guidance on the development and validation of diagnostic tests that depend on nucleic acid amplification and detection.

In order to comply with national and international clinical laboratory accreditation standards (e.g. the ISO 15189, Clinical Pathology Accreditation standards) and with the joint code of practice for research, there must be a method of assessing that test methods are "fit for purpose". This document gives guidance on development and describes how a validation file is produced. A test method may be a commercial kit, an in-house assay or reagent or a set of reagents bought separately and used to prepare an in house assay. A validation file is needed for both current and new test procedures. The file may refer to data recorded in workbooks, papers and reports. Modifications to assays (including commercially available assays) necessitate either an update to the validation file or creation of a new file. This paper is intended to provide a generic framework for in-house assay development and validation of new nucleic acid amplification assays including real-time polymerase chain reaction (PCR).

Nasal self-swabbing for estimating the prevalence of Staphylococcus aureus in the community.

Staphylococcus aureus remains a significant cause of morbidity and mortality and, therefore, a burden on healthcare systems. Our aim was to estimate the current rate of nasal S. aureus carriage in the general population and to determine the feasibility of nasal self-swabbing as a means of detection. Two thousand people (1200 adults and 800 children) from a single NHS general practice in Southampton, UK, were randomly selected from a general practice age sex register, stratified by age and sex, and invited to undertake nasal self-swabbing in their own home. Overall, 362 (32.5%) swabs from adults and 168 (22%) from children were returned. Responses were greater for adults and those of increased age, female gender and decreasing socio-economic deprivation. The overall estimated practice carriage rate of S. aureus directly standardized for age sex was 28% [95% confidence interval (CI) 26.1-30.2%]. Carriage of meticillin-susceptible S. aureus was 27% (95% CI 26.1-30.2%), whilst that of meticillin-resistant S. aureus was 1.9% (95% CI 0.7-3.1%). Although nasal self-swabbing rates were relatively low, they are comparable to other studies and may allow large population-based carriage studies to be undertaken at relatively low cost. Importantly, this study updates prevalence data for S. aureus carriage in the community.

Genotyping markers used for multi locus VNTR analysis with ompA (MLVA-ompA) and multi sequence typing (MST) retain stability in Chlamydia trachomatis.

We aimed to evaluate the stability of the Chlamydia trachomatis multi locus VNTR analysis (MLVA-ompA) and multi sequence typing (MST) systems through multiple passages in tissue culture. Firstly, we analyzed the stability of these markers through adaptation of C. trachomatis to tissue culture and secondly, we examined the stability of a four-locus MLVA-ompA and a five-locus MST system after multiple passages in tissue culture. Marker sequences were monitored through successive chlamydial developmental cycles to evaluate the stability of the individual DNA markers through many bacterial divisions and this, in turn, informed us of the usefulness of using such typing systems for short and long-term molecular epidemiology. Southampton genitourinary medicine (GUM) clinic isolates from endocervical swabs collected from C. trachomatis positive women were passaged through tissue culture. MLVA-ompA typing was applied to primary swab samples and to the same samples after C. trachomatis had been passaged through cell culture (eight passages). Sequence data from time-zero and passage-eight isolates were aligned with reference sequences to determine the stability of the markers. The Swedish new variant (nvCT) underwent 72 passages in cell culture and the markers of the two schemes were similarly analyzed. Analysis of genetic markers of the MLVA-ompA typing system before and after the isolates were introduced to tissue culture showed no change in the dominant sequence. The nvCT that had been passaged 72 times over the duration of a year also showed no variation in the dominant sequence for both the genotyping schemes. MLVA-ompA and MST markers are stable upon adaptation of C. trachomatis to tissue culture following isolation of strains from primary endocervical swab samples. These markers remain stable throughout multiple rounds of cell-division in tissue culture, concomitant with the incubation period and appearance of symptoms normally associated with host-infection. Both genotyping schemes are, therefore, suitable for epidemiology of C. trachomatis.

Long-term cultivation-independent microbial diversity analysis demonstrates that bacterial communities infecting the adult cystic fibrosis lung show stability and resilience.

BACKGROUND: Culture-independent analysis of the respiratory secretions of people with cystic fibrosis (CF) has identified many bacterial species not previously detected using culture in this context. However, little is known about their clinical significance or persistence in CF airways. METHODS: The authors characterised the viable bacterial communities in the sputum collected from 14 patients at monthly intervals over 1 year using a molecular community profiling technique-terminal restriction fragment length polymorphism. Clinical characteristics were also collected, including lung function and medications. Ecological community measures were determined for each sample. Microbial community change over time within subjects was defined using ecological analytical tools, and these measures were compared between subjects and to clinical features. RESULTS: Bacterial communities were stable within subjects over time but varied between subjects, despite similarities in clinical course. Antibiotic therapy temporarily perturbed these communities which generally returned to pretreatment configurations within 1 month. Species usually considered CF pathogens and those not previously regarded as such exhibited similar patterns of persistence. Less diverse sputum bacterial communities were correlated to lung disease severity and relative abundance of Pseudomonas aeruginosa. CONCLUSION: Whilst not true in all cases, the microbial communities that chronically infect the airways of patients with CF can vary little over a year despite antibiotic perturbation. The species present tended to vary more between than within subjects, suggesting that each CF airway infection is unique, with relatively stable and resilient bacterial communities. The inverse relationship between community richness and disease severity is similar to findings reported in other mucosal infections.