John Paul

Prediction of Staphylococcus aureus Antimicrobial Resistance by Whole-Genome Sequencing

ABSTRACT Whole-genome sequencing (WGS) could potentially provide a single platform for extracting all the information required to predict an organisms phenotype. However, its ability to provide accurate predictions has not yet been demonstrated in large independent studies of specific organisms. In this study, we aimed to develop a genotypic prediction method for antimicrobial susceptibilities. The whole genomes of 501 unrelated Staphylococcus aureus isolates were sequenced, and the assembled genomes were interrogated using BLASTn for a panel of known resistance determinants (chromosomal mutations and genes carried on plasmids). Results were compared with phenotypic susceptibility testing for 12 commonly used antimicrobial agents (penicillin, methicillin, erythromycin, clindamycin, tetracycline, ciprofloxacin, vancomycin, trimethoprim, gentamicin, fusidic acid, rifampin, and mupirocin) performed by the routine clinical laboratory. We investigated discrepancies by repeat susceptibility testing and manual inspection of the sequences and used this information to optimize the resistance determinant panel and BLASTn algorithm. We then tested performance of the optimized tool in an independent validation set of 491 unrelated isolates, with phenotypic results obtained in duplicate by automated broth dilution (BD Phoenix) and disc diffusion. In the validation set, the overall sensitivity and specificity of the genomic prediction method were 0.97 (95% confidence interval [95% CI], 0.95 to 0.98) and 0.99 (95% CI, 0.99 to 1), respectively, compared to standard susceptibility testing methods. The very major error rate was 0.5%, and the major error rate was 0.7%. WGS was as sensitive and specific as routine antimicrobial susceptibility testing methods. WGS is a promising alternative to culture methods for resistance prediction in S. aureus and ultimately other major bacterial pathogens.

Rapid, comprehensive, and affordable mycobacterial diagnosis with whole-genome sequencing: a prospective study

Summary Background Slow and cumbersome laboratory diagnostics for Mycobacterium tuberculosis complex (MTBC) risk delayed treatment and poor patient outcomes. Whole-genome sequencing (WGS) could potentially provide a rapid and comprehensive diagnostic solution. In this prospective study, we compare real-time WGS with routine MTBC diagnostic workflows. Methods We compared sequencing mycobacteria from all newly positive liquid cultures with routine laboratory diagnostic workflows across eight laboratories in Europe and North America for diagnostic accuracy, processing times, and cost between Sept 6, 2013, and April 14, 2014. We sequenced specimens once using local Illumina MiSeq platforms and processed data centrally using a semi-automated bioinformatics pipeline. We identified species or complex using gene presence or absence, predicted drug susceptibilities from resistance-conferring mutations identified from reference-mapped MTBC genomes, and calculated genetic distance to previously sequenced UK MTBC isolates to detect outbreaks. WGS data processing and analysis was done by staff masked to routine reference laboratory and clinical results. We also did a microcosting analysis to assess the financial viability of WGS-based diagnostics. Findings Compared with routine results, WGS predicted species with 93% (95% CI 90–96; 322 of 345 specimens; 356 mycobacteria specimens submitted) accuracy and drug susceptibility also with 93% (91–95; 628 of 672 specimens; 168 MTBC specimens identified) accuracy, with one sequencing attempt. WGS linked 15 (16% [95% CI 10–26]) of 91 UK patients to an outbreak. WGS diagnosed a case of multidrug-resistant tuberculosis before routine diagnosis was completed and discovered a new multidrug-resistant tuberculosis cluster. Full WGS diagnostics could be generated in a median of 9 days (IQR 6–10), a median of 21 days (IQR 14–32) faster than final reference laboratory reports were produced (median of 31 days [IQR 21–44]), at a cost of £481 per culture-positive specimen, whereas routine diagnosis costs £518, equating to a WGS-based diagnosis cost that is 7% cheaper annually than are present diagnostic workflows. Interpretation We have shown that WGS has a scalable, rapid turnaround, and is a financially feasible method for full MTBC diagnostics. Continued improvements to mycobacterial processing, bioinformatics, and analysis will improve the accuracy, speed, and scope of WGS-based diagnosis. Funding National Institute for Health Research, Department of Health, Wellcome Trust, British Colombia Centre for Disease Control Foundation for Population and Public Health, Department of Clinical Microbiology, Trinity College Dublin.

Whole-Genome Sequencing Shows That Patient-to-Patient Transmission Rarely Accounts for Acquisition of Staphylococcus aureus in an Intensive Care Unit

An assessment of Staphylococcus aureus acquisition among intensive care patients using serial sampling and whole-genome sequencing found less than a fifth of acquisitions resulted from patient-to-patient transmission. Whole-genome sequencing identified transmission more accurately than spa-typing and patient stay data.

Is infestation with the common bedbug increasing

EDITOR—In recent decades the common bedbug, Cimex lectularius , has been so scarce in the United Kingdom that new finds have been considered worthy of publication.1 In 1998 specimens from only one infestation were submitted to Brighton Public Health Laboratory Service for identification, none having been submitted during the previous three years. From February to October 1999specimens from four separate infestations were referred to the service; this suggests that bedbugs are becoming more common. Interestingly, in …

Reduction of Invasive Pneumococcal Disease 3 Years After the Introduction of the 13-Valent Conjugate Vaccine in the Oxfordshire Region of England

BACKGROUND The 7-valent pneumococcal conjugate (PCV7) vaccines impact on invasive pneumococcal disease (IPD) is well described, but few reports exist on the additional impact of the 13-valent vaccine (PCV13). METHODS We calculated the IPD incidence across all ages in a surveillance project following implementation of PCV7 (in September 2006) and PCV13 (in April 2010) in children aged <2 years (11 hospitals; 4935 cases). RESULTS The overall incidence decreased from 10 cases/100 000 persons per year in 1996-1997 to 8 cases/100 000 persons per year in 2007-2008 and 7 cases/100 000 in 2012-2013. Declines were greater in children aged <2 years (from 37 cases/100 000 in 1996-1997 to 29 and 14 cases/100 000 in 2007-2008 and 2012-2013, respectively). The incidence of IPD due to PCV7 serotypes decreased in all ages after PCV7 introduction (P < .001), whereas the incidence of IPD due to the additional 6 serotypes in PCV13 and to nonvaccine types (NVTs) increased in children aged ≥2 years (P < .001 for both comparisons). The incidence of IPD due to the 6 additional serotypes in PCV13 declined significantly after PCV13 introduction in all ages (P ≤ .01), and the incidence of IPD due to NVTs declined significantly in children aged ≥2 years (P = .003). In 2011-2013, the overall incidences of IPD due to PCV7 serotypes, the 6 additional serotypes in PCV13, and NVTs were 0.3, 2.8, and 4.4 cases/100 000; the incidences among children aged <2 years were 0.9, 2.4, and 10.8 cases/100 000, respectively. CONCLUSIONS The annual incidence of IPD due to vaccine serotypes (1-3 cases/100 000) among children aged <2 years and nontarget groups demonstrates the success of PCV7 and PCV13. A substantially higher incidence of IPD due to NVTs indicates the importance of ongoing surveillance and extension of vaccine polyvalency.

Population biology of Streptococcus pneumoniae isolated from oropharyngeal carriage and invasive disease

The population structure of Streptococcus pneumoniae in a sample of 134 carried antibiotic-susceptible isolates, and 53 resistant and susceptible invasive isolates, was examined using a DNA-based version of multilocus enzyme electrophoresis: multilocus restriction typing (MLRT). This involved RFLP analysis of PCR products generated from nine loci of housekeeping genes located around the pneumococcal chromosome. The combination of alleles at each of the nine loci gave an allelic profile or restriction type (RT). All carried (throat or nasopharyngeal) isolates from children or adults in Oxford and Manchester, UK, and from an HIV-seropositive cohort in Nairobi, Kenya, showed an epidemic population structure. Twelve carried clonal groups, each with different serotypes, were identified at both locations within the UK. Almost all of the carried clones examined (16/17) were found to possess identical RTs or sequence types (STs) to invasive isolates, indicating that frequently carried clones are also associated with cases of invasive disease. As expected from previous studies, the population of 53 invasive, mainly penicillin-resistant, isolates was also found to be at linkage equilibrium. Serotype switching was identified among 14% of RTs that possessed two or more members, or 5.7% of individual isolates within these RTs. In support of a population structure in which there is frequent recombination, there is also clear evidence that the trpA/B locus within pneumococci has evolved by horizontal gene transfer. A non-serotypable isolate from an HIV-seropositive patient in Kenya was clearly genetically distinct from other strains studied, with unique alleles at eight out of nine loci examined. However, it was initially identified as a pneumococcus by a 16S RNA gene probe (Gen-Probe), optochin susceptibility and the presence of pneumolysin and autolysin.

Recent transmission of tuberculosis in a cohort of HIV-1-infected female sex workers in Nairobi, Kenya

Objectives: To describe the epidemiological and clinical characteristics of HIV‐related tuberculosis in a female cohort, and to investigate the relative importance of recently transmitted infection and reactivation in the pathogenesis of adult HIV‐related tuberculosis. Design: Members of an established cohort of female sex workers in Nairobi were enrolled in a prospective study. Women were followed up regularly and seen on demand when sick. Methods: Between October 1989 and September 1992 we followed 587 HIV‐infected and 132 HIV‐seronegative women. Standard protocols were used to investigate common presentations. Cases of tuberculosis were identified clinically or by culture. All available Mycobacterium tuberculosis strains underwent DNA fingerprint analysis. Results: Forty‐nine incident and four recurrent episodes of tuberculosis were seen in HIV‐infected women; no disease was seen in seronegative sex workers (P = 0.0003). The overall incidence rate of tuberculosis was 34.5 per 1000 person‐years amongst HIV‐infected participants. In purified protein derivative (PPD) skin test‐positive women the rate was 66.7 per 1000 person‐years versus 18.1 per 1000 person‐years in PPD‐negative women. Twenty incident cases (41%) were clinically compatible with primary disease. DNA fingerprint analysis of strains from 32 incident cases identified two clusters comprising two and nine patients; allowing for index cases, 10 patients (28%) may have had recently transmitted disease. Three out of 10 (30%) patients who were initially PPD skin test‐negative became PPD‐positive. Taken together, 26 incident cases (53%) may have been recently infected. DNA fingerprint analysis also identified two (50%) of the four recurrent tuberculosis episodes as reinfection. Conclusions: Substantial recent transmission of tuberculosis appears to be occurring in Nairobi amongst HIV‐infected sex workers. It may be incorrect to assume in other regions of high tuberculosis transmission that active HIV‐related tuberculosis usually represents reactivation of latent infection.

Whole genome sequencing in the prevention and control of Staphylococcus aureus infection

Summary Background Staphylococcus aureus remains a leading cause of hospital-acquired infection but weaknesses inherent in currently available typing methods impede effective infection prevention and control. The high resolution offered by whole genome sequencing has the potential to revolutionise our understanding and management of S. aureus infection. Aim To outline the practicalities of whole genome sequencing and discuss how it might shape future infection control practice. Methods We review conventional typing methods and compare these with the potential offered by whole genome sequencing. Findings In contrast with conventional methods, whole genome sequencing discriminates down to single nucleotide differences and allows accurate characterisation of transmission events and outbreaks and additionally provides information about the genetic basis of phenotypic characteristics, including antibiotic susceptibility and virulence. However, translating its potential into routine practice will depend on affordability, acceptable turnaround times and on creating a reliable standardised bioinformatic infrastructure. Conclusion Whole genome sequencing has the potential to provide a universal test that facilitates outbreak investigation, enables the detection of emerging strains and predicts their clinical importance.

Waves of trouble: MRSA strain dynamics and assessment of the impact of infection control

There has been a sustained decline in bloodstream infections due to methicillin-resistant Staphylococcus aureus (MRSA) throughout the UK. The UK MRSA epidemic, which began in the 1990s, has been dominated by two epidemic MRSA (EMRSA) clones {EMRSA-15, of clonal complex (CC) 22 [sequence type (ST) 22], and EMRSA-16, of CC30 (ST36)}. It appears that both these clones followed a wave trajectory (initial expansion, relative stasis, then decline). Three recent studies have shown that ST36 has declined faster than ST22, a change that appears to have begun before the recent intensification of intensive control measures in the UK. The biological basis of infectious disease waves, including those of MRSA, is discussed, as are the implications of such waves for the assessment of the impact of infection control measures.

Whole-genome sequencing to determine transmission of Neisseria gonorrhoeae: an observational study

BACKGROUND New approaches are urgently required to address increasing rates of gonorrhoea and the emergence and global spread of antibiotic-resistant Neisseria gonorrhoeae. We used whole-genome sequencing to study transmission and track resistance in N gonorrhoeae isolates. METHODS We did whole-genome sequencing of isolates obtained from samples collected from patients attending sexual health services in Brighton, UK, between Jan 1, 2011, and March 9, 2015. We also included isolates from other UK locations, historical isolates from Brighton, and previous data from a US study. Samples from symptomatic patients and asymptomatic sexual health screening underwent nucleic acid amplification testing; positive samples and all samples from symptomatic patients were cultured for N gonorrhoeae, and resulting isolates were whole-genome sequenced. Cefixime susceptibility testing was done in selected isolates by agar incorporation, and we used sequence data to determine multi-antigen sequence types and penA genotypes. We derived a transmission nomogram to determine the plausibility of direct or indirect transmission between any two cases depending on the time between samples: estimated mutation rates, plus diversity noted within patients across anatomical sites and probable transmission pairs, were used to fit a coalescent model to determine the number of single nucleotide polymorphisms expected. FINDINGS 1407 (98%) of 1437 Brighton isolates between Jan 1, 2011, and March 9, 2015 were successfully sequenced. We identified 1061 infections from 907 patients. 281 (26%) of these infections were indistinguishable (ie, differed by zero single nucleotide polymorphisms) from one or more previous cases, and 786 (74%) had evidence of a sampled direct or indirect Brighton source. We observed multiple related samples across geographical locations. Of 1273 infections in Brighton (including historical data), 225 (18%) were linked to another case elsewhere in the UK, and 115 (9%) to a case in the USA. Four lineages initially identified in Brighton could be linked to 70 USA sequences, including 61 from a lineage carrying the mosaic penA XXXIV allele, which is associated with reduced cefixime susceptibility. INTERPRETATION We present a whole-genome-sequencing-based tool for genomic contact tracing of N gonorrhoeae and demonstrate local, national, and international transmission. Whole-genome sequencing can be applied across geographical boundaries to investigate gonorrhoea transmission and to track antimicrobial resistance. FUNDING Oxford National Institute for Health Research Health Protection Research Unit and Biomedical Research Centre.