Surveillance of Gram-negative bacteria: impact of variation in current European laboratory reporting practice on apparent multidrug resistance prevalence in paediatric bloodstream isolates.

This study evaluates whether estimated multidrug resistance (MDR) levels are dependent on the design of the surveillance system when using routine microbiological data. We used antimicrobial resistance data from the Antibiotic Resistance and Prescribing in European Children (ARPEC) project. The MDR status of bloodstream isolates of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa was defined using European Centre for Disease Prevention and Control (ECDC)-endorsed standardised algorithms (non-susceptible to at least one agent in three or more antibiotic classes). Assessment of MDR status was based on specified combinations of antibiotic classes reportable as part of routine surveillance activities. The agreement between MDR status and resistance to specific pathogen-antibiotic class combinations (PACCs) was assessed. Based on all available antibiotic susceptibility testing, the proportion of MDR isolates was 31% for E. coli, 30% for K. pneumoniae and 28% for P. aeruginosa isolates. These proportions fell to 9, 14 and 25%, respectively, when based only on classes collected by current ECDC surveillance methods. Resistance percentages for specific PACCs were lower compared with MDR percentages, except for P. aeruginosa. Accordingly, MDR detection based on these had low sensitivity for E. coli (2-41%) and K. pneumoniae (21-85%). Estimates of MDR percentages for Gram-negative bacteria are strongly influenced by the antibiotic classes reported. When a complete set of results requested by the algorithm is not available, inclusion of classes frequently tested as part of routine clinical care greatly improves the detection of MDR. Resistance to individual PACCs should not be considered reflective of MDR percentages in Enterobacteriaceae

Single-Nucleotide Polymorphism Genotyping Identifies a Locally Endemic Clone of Methicillin-Resistant Staphylococcus aureus

We developed, tested, and applied a TaqMan real-time PCR assay for interrogation of three single-nucleotide polymorphisms that differentiate a clade (termed ‘t003-X’) within the radiation of methicillin-resistant Staphylococcus aureus (MRSA) ST225. The TaqMan assay achieved 98% typeability and results were fully concordant with DNA sequencing. By applying this assay to 305 ST225 isolates from an international collection, we demonstrate that clade t003-X is endemic in a single acute-care hospital in Germany at least since 2006, where it has caused a substantial proportion of infections. The strain was also detected in another hospital located 16 kilometers away. Strikingly, however, clade t003-X was not found in 62 other hospitals throughout Germany nor among isolates from other countries, and, hence, displayed a very restricted geographical distribution. Consequently, our results show that SNP-typing may be useful to identify and track MRSA clones that are specific to individual healthcare institutions. In contrast, the spatial dissemination pattern observed here had not been resolved by other typing procedures, including multilocus sequence typing (MLST), spa typing, DNA macrorestriction, and multilocus variable-number tandem repeat analysis (MLVA)

Antibiotic Transport in Resistant Bacteria: Synchrotron UV Fluorescence Microscopy to Determine Antibiotic Accumulation with Single Cell Resolution

A molecular definition of the mechanism conferring bacterial multidrug resistance is clinically crucial and today methods for quantitative determination of the uptake of antimicrobial agents with single cell resolution are missing. Using the naturally occurring fluorescence of antibacterial agents after deep ultraviolet (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to separate the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic analysis, the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clinical antibitiotic could be determined and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is especially important to follow the behavior of quinolone molecules at individual cell level, to quantify the intracellular concentration of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addition, this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack

Quantifying Type-Specific Reproduction Numbers for Nosocomial Pathogens: Evidence for Heightened Transmission of an Asian Sequence Type 239 MRSA Clone

An important determinant of a pathogen's success is the rate at which it is transmitted from infected to susceptible hosts. Although there are anecdotal reports that methicillin-resistant Staphylococcus aureus (MRSA) clones vary in their transmissibility in hospital settings, attempts to quantify such variation are lacking for common subtypes, as are methods for addressing this question using routinely-collected MRSA screening data in endemic settings. Here we present a method to quantify the time-varying transmissibility of different subtypes of common bacterial nosocomial pathogens using routine surveillance data. The method adapts approaches for estimating reproduction numbers based on the probabilistic reconstruction of epidemic trees, but uses relative hazards rather than serial intervals to assign probabilities to different sources for observed transmission events. The method is applied to data collected as part of a retrospective observational study of a concurrent MRSA outbreak in the United Kingdom with dominant endemic MRSA clones (ST22 and ST36) and an Asian ST239 MRSA strain (ST239-TW) in two linked adult intensive care units, and compared with an approach based on a fully parametric transmission model. The results provide support for the hypothesis that the clones responded differently to an infection control measure based on the use of topical antiseptics, which was more effective at reducing transmission of endemic clones. They also suggest that in one of the two ICUs patients colonized or infected with the ST239-TW MRSA clone had consistently higher risks of transmitting MRSA to patients free of MRSA. These findings represent some of the first quantitative evidence of enhanced transmissibility of a pandemic MRSA lineage, and highlight the potential value of tailoring hospital infection control measures to specific pathogen subtypes

A Research Agenda for Helminth Diseases of Humans: Modelling for Control and Elimination

Mathematical modelling of helminth infections has the potential to inform policy and guide research for the control and elimination of human helminthiases. However, this potential, unlike in other parasitic and infectious diseases, has yet to be realised. To place contemporary efforts in a historical context, a summary of the development of mathematical models for helminthiases is presented. These efforts are discussed according to the role that models can play in furthering our understanding of parasite population biology and transmission dynamics, and the effect on such dynamics of control interventions, as well as in enabling estimation of directly unobservable parameters, exploration of transmission breakpoints, and investigation of evolutionary outcomes of control. The Disease Reference Group on Helminth Infections (DRG4), established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR), was given the mandate to review helminthiases research and identify research priorities and gaps. A research and development agenda for helminthiasis modelling is proposed based on identified gaps that need to be addressed for models to become useful decision tools that can support research and control operations effectively. This agenda includes the use of models to estimate the impact of large-scale interventions on infection incidence; the design of sampling protocols for the monitoring and evaluation of integrated control programmes; the modelling of co-infections; the investigation of the dynamical relationship between infection and morbidity indicators; the improvement of analytical methods for the quantification of anthelmintic efficacy and resistance; the determination of programme endpoints; the linking of dynamical helminth models with helminth geostatistical mapping; and the investigation of the impact of climate change on human helminthiases. It is concluded that modelling should be embedded in helminth research, and in the planning, evaluation, and surveillance of interventions from the outset. Modellers should be essential members of interdisciplinary teams, propitiating a continuous dialogue with end users and stakeholders to reflect public health needs in the terrain, discuss the scope and limitations of models, and update biological assumptions and model outputs regularly. It is highlighted that to reach these goals, a collaborative framework must be developed for the collation, annotation, and sharing of databases from large-scale anthelmintic control programmes, and that helminth modellers should join efforts to tackle key questions in helminth epidemiology and control through the sharing of such databases, and by using diverse, yet complementary, modelling approaches