The UKMYC5 plate is a 96-well microtiter plate designed by the CRyPTIC Consortium (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium) to enable the measurement of MICs ...of 14 different antituberculosis (anti-TB) compounds for >30,000 clinical
isolates. Unlike the MYCOTB plate, on which the UKMYC5 plate is based, the UKMYC5 plate includes two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds. UKMYC5 plates were tested by seven laboratories on four continents by use of a panel of 19 external quality assessment (EQA) strains, including H37Rv. To assess the optimal combination of reading method and incubation time, MICs were measured from each plate by two readers, using three methods (mirrored box, microscope, and Vizion digital viewing system), after 7, 10, 14, and 21 days of incubation. In addition, all EQA strains were subjected to whole-genome sequencing and phenotypically characterized by the 7H10/7H11 agar proportion method (APM) and by use of MGIT960 mycobacterial growth indicator tubes. We concluded that the UKMYC5 plate is optimally read using the Vizion system after 14 days of incubation, achieving an interreader agreement of 97.9% and intra- and interlaboratory reproducibility rates of 95.6% and 93.1%, respectively. The mirrored box had a similar reproducibility. Strains classified as resistant by APM, MGIT960, or the presence of mutations known to confer resistance consistently showed elevated MICs compared to those for strains classified as susceptible. Finally, the UKMYC5 plate records intermediate MICs for one strain for which the APM measured MICs close to the applied critical concentration, providing early evidence that the UKMYC5 plate can quantitatively measure the magnitude of resistance to anti-TB compounds that is due to specific genetic variation.
Staphylococcus aureus is a major cause of healthcare associated mortality, but like many important bacterial pathogens, it is a common constituent of the normal human body flora. Around a third of ...healthy adults are carriers. Recent evidence suggests that evolution of S. aureus during nasal carriage may be associated with progression to invasive disease. However, a more detailed understanding of within-host evolution under natural conditions is required to appreciate the evolutionary and mechanistic reasons why commensal bacteria such as S. aureus cause disease. Therefore we examined in detail the evolutionary dynamics of normal, asymptomatic carriage. Sequencing a total of 131 genomes across 13 singly colonized hosts using the Illumina platform, we investigated diversity, selection, population dynamics and transmission during the short-term evolution of S. aureus.
We characterized the processes by which the raw material for evolution is generated: micro-mutation (point mutation and small insertions/deletions), macro-mutation (large insertions/deletions) and the loss or acquisition of mobile elements (plasmids and bacteriophages). Through an analysis of synonymous, non-synonymous and intergenic mutations we discovered a fitness landscape dominated by purifying selection, with rare examples of adaptive change in genes encoding surface-anchored proteins and an enterotoxin. We found evidence for dramatic, hundred-fold fluctuations in the size of the within-host population over time, which we related to the cycle of colonization and clearance. Using a newly-developed population genetics approach to detect recent transmission among hosts, we revealed evidence for recent transmission between some of our subjects, including a husband and wife both carrying populations of methicillin-resistant S. aureus (MRSA).
This investigation begins to paint a picture of the within-host evolution of an important bacterial pathogen during its prevailing natural state, asymptomatic carriage. These results also have wider significance as a benchmark for future systematic studies of evolution during invasive S. aureus disease.
Summary Background Health-care workers have been implicated in nosocomial outbreaks of Staphylococcus aureus , but the dearth of evidence from non-outbreak situations means that routine health-care ...worker screening and S aureus eradication are controversial. We aimed to determine how often S aureus is transmitted from health-care workers or the environment to patients in an intensive care unit (ICU) and a high-dependency unit (HDU) where standard infection control measures were in place. Methods In this longitudinal cohort study, we systematically sampled health-care workers, the environment, and patients over 14 months at the ICU and HDU of the Royal Sussex County Hospital, Brighton, England. Nasal swabs were taken from health-care workers every 4 weeks, bed spaces were sampled monthly, and screening swabs were obtained from patients at admission to the ICU or HDU, weekly thereafter, and at discharge. Isolates were cultured and their whole genome sequenced, and we used the threshold of 40 single-nucleotide variants (SNVs) or fewer to define subtypes and infer recent transmission. Findings Between Oct 31, 2011, and Dec 23, 2012, we sampled 198 health-care workers, 40 environmental locations, and 1854 patients; 1819 isolates were sequenced. Median nasal carriage rate of S aureus in health-care workers at 4-weekly timepoints was 36·9% (IQR 35·7–37·3), and 115 (58%) health-care workers had S aureus detected at least once during the study. S aureus was identified in 8–50% of environmental samples. 605 genetically distinct subtypes were identified (median SNV difference 273, IQR 162–399) at a rate of 38 (IQR 34–42) per 4-weekly cycle. Only 25 instances of transmission to patients (seven from health-care workers, two from the environment, and 16 from other patients) were detected. Interpretation In the presence of standard infection control measures, health-care workers were infrequently sources of transmission to patients. S aureus epidemiology in the ICU and HDU is characterised by continuous ingress of distinct subtypes rather than transmission of genetically related strains. Funding UK Medical Research Council, Wellcome Trust, Biotechnology and Biological Sciences Research Council, UK National Institute for Health Research, and Public Health England.
In sub-Saharan Africa, bacterial meningitis is common and is associated with a high mortality. Adjuvant therapy with corticosteroids reduces mortality among adults in the developed world, but it has ...not been adequately tested in developing countries or in the context of advanced human immunodeficiency virus (HIV) infection.
We conducted a randomized, double-blind, placebo-controlled trial of dexamethasone (16 mg twice daily for 4 days) and an open-label trial of intramuscular versus intravenous ceftriaxone (2 g twice daily for 10 days) in adults with an admission diagnosis of bacterial meningitis in Blantyre, Malawi. The primary outcome was death at 40 days after randomization.
A total of 465 patients, 90% of whom were HIV-positive, were randomly assigned to receive dexamethasone (233 patients) or placebo (232 patients) plus intramuscular ceftriaxone (230 patients) or intravenous ceftriaxone (235 patients). There was no significant difference in mortality at 40 days in the corticosteroid group (129 of 231 patients) as compared with the placebo group (120 of 228 patients) by intention-to-treat analysis (odds ratio, 1.14; 95% confidence interval CI, 0.79 to 1.64) or when the analysis was restricted to patients with proven pneumococcal meningitis (68 of 129 patients receiving corticosteroids vs. 72 of 143 patients receiving placebo) (odds ratio, 1.10; 95% CI, 0.68 to 1.77). There were no significant differences between groups in the outcomes of disability and death combined, hearing impairment, and adverse events. There was no difference in mortality with intravenous ceftriaxone (121 of 230 patients) as compared with intramuscular ceftriaxone (128 of 229 patients) (odds ratio, 0.88; 95% CI, 0.61 to 1.27).
Adjuvant therapy with dexamethasone for bacterial meningitis in adults from an area with a high prevalence of HIV did not reduce mortality or morbidity. In this setting, intramuscular administration was not inferior to intravenous administration of ceftriaxone for bacterial meningitis. (Current Controlled Trials number, ISRCTN31371499 controlled-trials.com.).
Summary Background The control of Clostridium difficile infections is an international clinical challenge. The incidence of C difficile in England declined by roughly 80% after 2006, following the ...implementation of national control policies; we tested two hypotheses to investigate their role in this decline. First, if C difficile infection declines in England were driven by reductions in use of particular antibiotics, then incidence of C difficile infections caused by resistant isolates should decline faster than that caused by susceptible isolates across multiple genotypes. Second, if C difficile infection declines were driven by improvements in hospital infection control, then transmitted (secondary) cases should decline regardless of susceptibility. Methods Regional (Oxfordshire and Leeds, UK) and national data for the incidence of C difficile infections and antimicrobial prescribing data (1998–2014) were combined with whole genome sequences from 4045 national and international C difficile isolates. Genotype (multilocus sequence type) and fluoroquinolone susceptibility were determined from whole genome sequences. The incidence of C difficile infections caused by fluoroquinolone-resistant and fluoroquinolone-susceptible isolates was estimated with negative-binomial regression, overall and per genotype. Selection and transmission were investigated with phylogenetic analyses. Findings National fluoroquinolone and cephalosporin prescribing correlated highly with incidence of C difficile infections (cross-correlations >0·88), by contrast with total antibiotic prescribing (cross-correlations <0·59). Regionally, C difficile decline was driven by elimination of fluoroquinolone-resistant isolates (approximately 67% of Oxfordshire infections in September, 2006, falling to approximately 3% in February, 2013; annual incidence rate ratio 0·52, 95% CI 0·48–0·56 vs fluoroquinolone-susceptible isolates: 1·02, 0·97–1·08). C difficile infections caused by fluoroquinolone-resistant isolates declined in four distinct genotypes (p<0·01). The regions of phylogenies containing fluoroquinolone-resistant isolates were short-branched and geographically structured, consistent with selection and rapid transmission. The importance of fluoroquinolone restriction over infection control was shown by significant declines in inferred secondary (transmitted) cases caused by fluoroquinolone-resistant isolates with or without hospital contact (p<0·0001) versus no change in either group of cases caused by fluoroquinolone-susceptible isolates (p>0·2). Interpretation Restricting fluoroquinolone prescribing appears to explain the decline in incidence of C difficile infections, above other measures, in Oxfordshire and Leeds, England. Antimicrobial stewardship should be a central component of C difficile infection control programmes. Funding UK Clinical Research Collaboration (Medical Research Council, Wellcome Trust, National Institute for Health Research); NIHR Oxford Biomedical Research Centre; NIHR Health Protection Research Unit on Healthcare Associated Infection and Antimicrobial Resistance (Oxford University in partnership with Public Health England PHE), and on Modelling Methodology (Imperial College, London in partnership with PHE); and the Health Innovation Challenge Fund.
Extensive global sampling and sequencing of the pandemic virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have enabled researchers to monitor its spread and to identify concerning ...new variants. Two important determinants of variant spread are how frequently they arise within individuals and how likely they are to be transmitted. To characterize within-host diversity and transmission, we deep-sequenced 1313 clinical samples from the United Kingdom. SARS-CoV-2 infections are characterized by low levels of within-host diversity when viral loads are high and by a narrow bottleneck at transmission. Most variants are either lost or occasionally fixed at the point of transmission, with minimal persistence of shared diversity, patterns that are readily observable on the phylogenetic tree. Our results suggest that transmission-enhancing and/or immune-escape SARS-CoV-2 variants are likely to arise infrequently but could spread rapidly if successfully transmitted.
Over the last 20 years there have been 32 reports of carbapenem-resistant organisms in the hospital water environment, with half of these occurring since 2010. The majority of these reports have ...described associated clinical outbreaks in the intensive care setting, affecting the critically ill and the immunocompromised. Drains, sinks, and faucets were most frequently colonized, and Pseudomonas aeruginosa the predominant organism. Imipenemase (IMP), Klebsiella pneumoniae carbapenemase (KPC), and Verona integron-encoded metallo-β-lactamase (VIM) were the most common carbapenemases found. Molecular typing was performed in almost all studies, with pulse field gel electrophoresis being most commonly used. Seventy-two percent of studies reported controlling outbreaks, of which just more than one-third eliminated the organism from the water environment. A combination of interventions seems to be most successful, including reinforcement of general infection control measures, alongside chemical disinfection. The most appropriate disinfection method remains unclear, however, and it is likely that replacement of colonized water reservoirs may be required for long-term clearance.
Two recently completed phase 3 trials (003 and 004) showed fidaxomicin to be noninferior to vancomycin for curing Clostridium difficile infection (CDI) and superior for reducing CDI recurrences. In ...both studies, adults with active CDI were randomized to receive blinded fidaxomicin 200 mg twice daily or vancomycin 125 mg 4 times a day for 10 days. Post hoc exploratory intent-to-treat (ITT) time-to-event analyses were undertaken on the combined study 003 and 004 data, using fixed-effects meta-analysis and Cox regression models. ITT analysis of the combined 003/004 data for 1164 patients showed that fidaxomicin reduced persistent diarrhea, recurrence, or death by 40% (95% confidence interval CI, 26%—51%; P < .0001) compared with vancomycin through day 40. A 37% (95% CI, 2%—60%; P = .037) reduction in persistent diarrhea or death was evident through day 12 (heterogeneity P = .50 vs 13—40 days), driven by 7 (1.2%) fidaxomicin versus 17 (2.9%) vancomycin deaths at <12 days. Low albumin level, low eosinophil count, and CDI treatment preenrollment were risk factors for persistent diarrhea or death at 12 days, and CDI in the previous 3 months was a risk factor for recurrence (all P < .01). Fidaxomicin has the potential to substantially improve outcomes from CDI.
Symptomatic recurrence of Clostridium difficile infection (CDI) occurs in approximately 20% of patients and is challenging to treat. Identifying those at high risk could allow targeted initial ...management and improve outcomes. Adult toxin enzyme immunoassay—positive CDI cases in a population of approximately 600 000 persons from September 2006 through December 2010 were combined with epidemiological/clinical data. The cumulative incidence of recurrence ≥14 days after the diagnosis and/or onset of first-ever CDI was estimated, treating death without recurrence as a competing risk, and predictors were identified from cause-specific proportional hazards regression models. A total of 1678 adults alive 14 days after their first CDI were included; median age was 77 years, and 1191 (78%) were inpatients. Of these, 363 (22%) experienced a recurrence ≥14 days after their first CDI, and 594 (35%) died without recurrence through March 2011. Recurrence risk was independently and significantly higher among patients admitted as emergencies, with previous gastrointestinal ward admission(s), last discharged 4—12 weeks before first diagnosis, and with CDI diagnosed at admission. Recurrence risk also increased with increasing age, previous total hours admitted, and C-reactive protein level at first CDI (all P < .05). The 4-month recurrence risk increased by approximately 5% (absolute) for every 1-point increase in a risk score based on these factors. Risk factors, including increasing age, initial disease severity, and hospital exposure, predict CDI recurrence and identify patients likely to benefit from enhanced initial CDI treatment.
Escherichia colisequence type 131 (ST131) has emerged globally as the most predominant extraintestinal pathogenic lineage within this clinically important species, and its association with ...fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined. This study of the largest worldwide collection (n= 215) of sequenced ST131E. coliisolates to date demonstrates that the clonal expansion of two previously recognized antimicrobial-resistant clades, C1/H30R and C2/H30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/H30Rx clade driven by the acquisition of ablaCTX-M-15-containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids and chromosomal integration ofblaCTX-Mwithin subclusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed among ST131, theblaCTX-M-14/14-likegroup. The complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages ofE. coli These data also highlight the global imperative to reduce specific antibiotic selection pressures and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages.
Escherichia coli, perennially a major bacterial pathogen, is becoming increasingly difficult to manage due to emerging resistance to all preferred antimicrobials. Resistance is concentrated within specificE. colilineages, such as sequence type 131 (ST131). Clarification of the genetic basis for clonally associated resistance is key to devising intervention strategies. We used high-resolution genomic analysis of a large global collection of ST131 isolates to define the evolutionary history of extended-spectrum beta-lactamase production in ST131. We documented diverse contributory genetic processes, including stable chromosomal integrations of resistance genes, persistence and evolution of mobile resistance elements within sublineages, and sporadic acquisition of different resistance elements. Both global distribution and regional segregation were evident. The diversity of resistance element acquisition and propagation within ST131 indicates a need for control and surveillance strategies that target both bacterial strains and mobile genetic elements.
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