Peter Marsh

Whole-genome analysis of diverse Chlamydia trachomatis strains identifies phylogenetic relationships masked by current clinical typing

Chlamydia trachomatis is responsible for both trachoma and sexually transmitted infections, causing substantial morbidity and economic cost globally. Despite this, our knowledge of its population and evolutionary genetics is limited. Here we present a detailed phylogeny based on whole-genome sequencing of representative strains of C. trachomatis from both trachoma and lymphogranuloma venereum (LGV) biovars from temporally and geographically diverse sources. Our analysis shows that predicting phylogenetic structure using ompA, which is traditionally used to classify Chlamydia, is misleading because extensive recombination in this region masks any true relationships present. We show that in many instances, ompA is a chimera that can be exchanged in part or as a whole both within and between biovars. We also provide evidence for exchange of, and recombination within, the cryptic plasmid, which is another key diagnostic target. We used our phylogenetic framework to show how genetic exchange has manifested itself in ocular, urogenital and LGV C. trachomatis strains, including the epidemic LGV serotype L2b.

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.

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.

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.

Does bacterial density in cystic fibrosis sputum increase prior to pulmonary exacerbation

BACKGROUND Cystic Fibrosis (CF) lung disease is characterised by an inexorable decline in lung function, punctuated by periods of symptomatic worsening known as pulmonary exacerbations (referred to here as CFPE). Despite their clinical significance, the cause of CFPE remains undetermined. It has been suggested that an increase in bacterial density may be a trigger, although this has not been shown empirically. METHODS Here, a previously validated quantitative PCR-based approach was used to assess numbers of Pseudomonas aeruginosa and of total bacteria in respiratory secretions from patients during the period leading up to CFPE. Sputum samples collected from 12 adult CF patients were selected retrospectively to fall approximately 21, 14, 7 and 0 days prior to CFPE diagnosis. In addition, the relationships between clinical parameters (FEV(1), temperature and patient reported outcome measures) and microbiological data were investigated. RESULTS No significant changes either in total bacterial or P. aeruginosa numbers were identified prior to CFPE. Of all the correlations tested, only temperature showed a significant correlation with total bacterial numbers in the period leading to CFPE. CONCLUSIONS These findings strongly suggest that CFPE do not generally result from increased bacterial density within the airways. Instead, data presented here are consistent with alternative models of pulmonary exacerbation.

The exclusion of dead bacterial cells is essential for accurate molecular analysis of clinical samples.

The DNA-based techniques used to detect bacteria in clinical samples are unable to discriminate between live bacteria, dead bacteria, and extracellular DNA. This failure to limit analysis to viable bacterial cells represents a significant problem, leading to false-positive results, as well as a failure to resolve the impact of antimicrobial therapy. The use of propidium monoazide treatment significantly reduces the contribution of dead cells and extracellular DNA to such culture-independent analyses. Here, the increased ability to resolve the impact of antibiotic therapy on Pseudomonas aeruginosa load in cystic fibrosis respiratory samples reveals statistically significant changes that would otherwise go undetected.

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.

Evaluation of BacLite Rapid MRSA, a rapid culture based screening test for the detection of ciprofloxacin and methicillin resistant S. aureus (MRSA) from screening swabs

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen worldwide. The need for accurate and rapid screening methods to detect MRSA carriers has been clearly established. The performance of a novel assay, BacLite Rapid MRSA (Acolyte Biomedica, UK) for the rapid detection (5 h) and identification of hospital associated ciprofloxacin resistant strains of MRSA directly from nasal swab specimens was compared to that obtained by culture on Mannitol salt agar containing Oxacillin (MSAO) after 48 h incubation.ResultsA total of 1382 nasal screening swabs were tested by multiple operators. The BacLite Rapid MRSA test detected 142 out of the 157 confirmed MRSA that were detected on MSAO giving a diagnostic sensitivity of 90.4, diagnostic specificity of 95.7% and a negative predictive value of 98.7%. Of the 15 false negatives obtained by the BacLite Rapid MRSA test, seven grew small amounts (< 10 colonies of MRSA) on the MSAO culture plate and five isolates were ciprofloxacin sensitive. However there were 13 confirmed BacLite MRSA positive samples, which were negative by the direct culture method, probably due to overgrowth on the MSAO plate. There were 53 false positive results obtained by the BacLite Rapid MRSA test at 5 h and 115 cases where MRSA colonies were tentatively identified on the MSAO plate when read at 48 h, and which subsequently proved not to be MRSA.ConclusionThe Baclite MRSA test is easy to use and provides a similar level of sensitivity to conventional culture for the detection of nasal carriage of MRSA with the advantage that the results are obtained much more rapidly.

Characterisation of bacteria in ascites—reporting the potential of culture-independent, molecular analysis

Spontaneous bacterial peritonitis (SBP) is a severe complication of liver disease. A significant proportion of patients have culture-negative ascites, despite having similar signs, symptoms and mortality to those with SBP. Therefore, empirical antibiotic treatment for infection is often started without knowledge of the causative organisms. Here, we investigated the potential of molecular techniques to provide rapid and accurate characterisation of the bacteria present in ascitic fluid. Ascites samples were obtained from 29 cirrhotic patients undergoing clinically indicated therapeutic paracentesis. Bacterial content was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis, quantitative polymerase chain reaction (PCR) and 16S ribosomal clone sequence analysis. Bacterial signal was detected in all samples, compared to three out of ten using standard methods. Bacterial loads ranged from 5.5 × 102 to 5.4 × 107 cfu/ml, with a mean value of 1.9 × 106 cfu/ml (standard deviation ± 9.6 × 106 cfu/ml). In all but one instance, bacterial species identified by culture were also confirmed by molecular analyses. Preliminary data presented here suggests that culture-independent, molecular analyses could provide rapid characterisation of the bacterial content of ascites fluid, providing a basis for the investigation of SBP development and allowing early and targeted antibiotic intervention.

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

Chlamydia trachomatis is the worlds 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 pathogens 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.