Multilocus sequence types of invasive Corynebacterium diphtheriae isolated in the Rio de Janeiro urban area, Brazil.

Invasive infections caused by Corynebacterium diphtheriae in vaccinated and non-vaccinated individuals have been reported increasingly. In this study we used multilocus sequence typing (MLST) to study genetic relationships between six invasive strains of this bacterium isolated solely in the urban area of Rio de Janeiro, Brazil, during a 10-year period. Of note, all the strains rendered negative results in PCR reactions for the tox gene, and four strains presented an atypical sucrose-fermenting ability. Five strains represented new sequence types. MLST results did not support the hypothesis that invasive (sucrose-positive) strains of C. diphtheriae are part of a single clonal complex. Instead, one of the main findings of the study was that such strains can be normally found in clonal complexes with strains related to non-invasive disease. Comparative analyses with C. diphtheriae isolated in different countries provided further information on the geographical circulation of some sequence types.

Waterline Disinfectants Reduce Dental Bioaerosols: A Multitracer Validation.

Oral microbes are dispersed during dental treatment and reduction methods have been proposed, but dental unit waterline (DUWL) disinfectants have received little attention; specifically, the effect on viruses has not been studied. This study aims to 1) investigate the effect of DUWL disinfectants on viral dispersion in dental bioaerosols and 2) establish a dual-tracer system using live bacteriophage and fluorescein supported by optical particle measurement. Bacteriophage MS2 was used as a viral tracer and fluorescein as a fluorescent tracer. Validation experiments were conducted to exclude interference of one tracer with the other or of DUWL disinfectants on detection methods. Simulated "saliva" containing the tracers was infused into the mouth of a dental mannequin during 10-min dental procedures with an air turbine handpiece (n = 3 replicates). Aerosols and droplets were sampled in an enclosed dental operatory using air samplers and settlement onto sterile filter papers. Bacteriophage was quantified using plaque assays and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Fluorescein was quantified fluorometrically. The effect of DUWL disinfectants on total aerosol concentration was assessed in separate experiments using an optical particle counter. DUWL disinfectants reduced bacteriophage viability, and interference between tracers was not observed. In simulated clinical procedures, the disinfectant ICX reduced bacteriophage detection substantially (P < 0.001; 2-way analysis of variance). MS2 RNA was detected in all experimental samples but not negative controls. Samples positive on RT-qPCR but not plaque assays may indicate that virions at distant sites are nonviable. Fluorescein tracer showed good agreement with the bacteriophage tracer. DUWL disinfectants designed for continuous presence in irrigants reduce the dispersion of viable virus in dental bioaerosols during simulated procedures. Their use may therefore be important for routine infection control and as a mitigation factor during infectious disease outbreaks. Future studies should explore this using a range of viruses and other microbes.

Local Exhaust Ventilation to Control Dental Aerosols and Droplets.

Dental procedures produce aerosols that may remain suspended and travel significant distances from the source. Dental aerosols and droplets contain oral microbes, and there is potential for infectious disease transmission and major disruption to dental services during infectious disease outbreaks. One method to control hazardous aerosols often used in industry is local exhaust ventilation (LEV). The aim of this study was to investigate the effect of LEV on aerosols and droplets produced during dental procedures. Experiments were conducted on dental mannequins in an 825.4-m3 open-plan clinic and a 49.3-m3 single surgery. Ten-minute crown preparations were performed with an air-turbine handpiece in the open-plan clinic and 10-min full-mouth ultrasonic scaling in the single surgery. Fluorescein was added to instrument irrigation reservoirs as a tracer. In both settings, optical particle counters (OPCs) were used to measure aerosol particles between 0.3 and 10.0 µm, and liquid cyclone air samplers were used to capture aerosolized fluorescein tracer. In addition, in the open-plan setting, fluorescein tracer was captured by passive settling onto filter papers in the environment. Tracer was quantified fluorometrically. An LEV device with high-efficiency particulate air filtration and a flow rate of 5,000 L/min was used. LEV reduced aerosol production from the air-turbine handpiece by 90% within 0.5 m, and this was 99% for the ultrasonic scaler. OPC particle counts were substantially reduced for both procedures and air-turbine settled droplet detection reduced by 95% within 0.5 m. The effect of LEV was substantially greater than suction alone for the air-turbine and was similar to the effect of suction for the ultrasonic scaler. LEV reduces aerosol and droplet contamination from dental procedures by at least 90% in the breathing zone of the operator, and it is therefore a valuable tool to reduce the dispersion of dental aerosols.

The safety and efficacy of botulinum toxin-A in the management of bladder oversensitivity: a randomised double-blind placebo-controlled trial.

OBJECTIVE: To assess the safety and efficacy of botulinum toxin-A (botn-A) in the management of patients with bladder oversensitivity (BO). PATIENTS AND METHODS: Twenty-three consecutive patients with a diagnosis of BO refractory to anticholinergics were enrolled in this randomised, double blind, placebo-controlled trial. Patients were randomly allocated to receive intradetrusor injections of either botn-A (100 U Botox) or saline (placebo) via a flexible cystoscopic approach. The study was designed to have 90% power to detect a change in the maximum cystometric capacity (MCC) of 30%. It was calculated that a total cohort of 58 patients would be required. Urodynamic assessment (UDS), voiding diaries (VD) and quality of life (QoL) were assessed at baseline and at 4 and 12 weeks following intervention. RESULTS: An interim analysis was performed and the trial halted after recruitment of 23 patients as a result of poorly perceived patient benefit. Data were analysed for 21 patients (10 botn-A; 11 placebo). In the treatment arm, there was a significant increase in MCC (mean rise 105 ml; p = 0.009). However, storage symptoms remained statistically unchanged following botn-A. Three patients in the treatment arm were required to perform clean intermittent self-catheterisation with no clinical improvement. The limitations of this trial include the small sample size and the unplanned interim analysis. CONCLUSIONS: This is the first randomised, double blind, placebo-controlled trial examining the effects of botn-A exclusively in patients with BO. A significant increase in MCC was observed but this did not translate to clinical benefit with no change observed in the symptoms and quality of life for the majority of patients.

To Push or To Pull? In a Post-COVID World, Supporting and Incentivizing Antimicrobial Drug Development Must Become a Governmental Priority.

The COVID-19 pandemic has refocused attention worldwide on the dangers of infectious diseases, in terms of both global health and the effects on the world economy. Even in high income countries, health systems have been found wanting in dealing with the new infectious agent. However, the even greater long-term danger of antimicrobial resistance in pathogenic bacteria and fungi is still under-appreciated, especially among the general public. Although antimicrobial drug development faces significant scientific challenges, the gravest challenge at the moment appears to be economic, where the lack of a viable market has led to a collapse in drug development pipelines. There is therefore a critical need for governments across the world to further incentivize the development of antimicrobials. Most incentive strategies over the past decade have focused on so-called "push" incentives that bridge the costs of antimicrobial research and development, but these have been insufficient for reviving the pipeline. In this Perspective, we analyze the current incentive strategies in place for antimicrobial drug development, and focus on "pull" incentives, which instead aim to improve revenue generation and thereby resolve the antimicrobial market failure challenge. We further analyze these incentives in a broader "One Health" context and stress the importance of developing and enforcing strict protocols to ensure appropriate manufacturing practices and responsible use. Our analysis reiterates the importance of international cooperation, coordination across antimicrobial research, and sustained funding in tackling this significant global challenge. A failure to invest wisely and continuously to incentivize antimicrobial pipelines will have catastrophic consequences for global health and wellbeing in the years to come.

Evaluation of implementation of risk management guidelines for carriers of pathogenic variants in mismatch repair genes: a nationwide audit of familial cancer clinics.

INTRODUCTION: This nationwide study assessed the impact of Lynch syndrome-related risk management guidelines on clinicians' recommendations of risk management strategies to carriers of pathogenic variants in mismatch repair genes and the extent to which carriers took up strategies in concordance with guidelines. MATERIALS AND METHODS: Clinic files of 464 carriers (with and without colorectal cancer) were audited for carriers who received their genetic testing results in July 2008-July 2009 (i.e. before guideline release), July 2010-July 2011 and July 2012-July 2013 (both after guideline release) at 12 familial cancer clinics (FCCs) to ascertain the extent to which carriers were informed about risk management in accordance with guidelines. All carriers captured by the audit were invited to participate in interviews; 215 were interviewed to assess adherence to recommended risk management guidelines. RESULTS: The rates of documentation in clinic files increased significantly from pre- to post-guideline for only two out of eight risk management strategies. The strategies with the highest compliance of carriers post-guidelines were: uptake of one or two-yearly colonoscopy (87%), followed by hysterectomy to prevent endometrial cancer (68%), aspirin as risk-reducing medication (67%) and risk-reducing salpingo-oophorectomy (63%). Interrater reliability check for all guidelines showed excellent agreement (k statistics = 0.89). CONCLUSION: These results indicate that there is scope to further increase provision of advice at FCCs to ensure that all carriers receive recommendations about evidence-based risk management. A multi-pronged behaviour change and implementation science approach tailored to specific barriers is likely to be needed to achieve optimal clinician behaviours and outcomes for carriers.

Experience gained by basic surgical trainees in varicose vein surgery over a 15 year period - has it changed?

OBJECTIVE: The aim of this study was to see if the training provided for Basic Surgical Trainees (BST's) by one consultant vascular surgeon has changed over a 15-year period. METHOD: From a computerized database we have a 15-year record of varicose vein operations identifying the first and second surgeon. We have analysed cases involving the BST and those in which the consultant operated alone. In such instances training opportunities were lost. RESULTS: No change was found in the total number of cases performed, the number of operations carried by the BST as the first surgeon, or the cases performed by the consultant operating alone. The number of operations performed by a BST annually correlated positively with the total number of cases. A BST was named as first surgeon in 39% of cases (632/1622). CONCLUSION: This study has shown no definite evidence to support the view that training in varicose vein surgery has deteriorated.

Burkholderia cepacia complex bacteria: opportunistic pathogens with important natural biology.

Interaction with plants around their roots and foliage forms the natural habitat for a wide range of gram-negative bacteria such as Burkholderia, Pseudomonas and Ralstonia. During these interactions many of these bacteria facilitate highly beneficial processes such as the breakdown of pollutants or enhancement of crop growth. All these bacterial species are also capable of causing opportunistic infections in vulnerable individuals, especially people with cystic fibrosis (CF). Here we will review the current understanding of the Burkholderia cepacia complex (Bcc) as a group of model opportunistic pathogens, contrasting their clinical epidemiology with their ecological importance. Currently, the B. cepacia complex is composed of nine formally named species groups which are all difficult to identify using phenotypic methods. Genetic methods such as 16S rRNA and recA gene sequence analysis have proven useful for Bcc species identification. Multilocus sequence typing (MLST) is also emerging as a very useful tool for both Bcc strain and species identification. Historically, Burkholderia cenocepacia was the most dominant Bcc pathogen in CF, however, probably as a result of strict infection control practices introduced to control the spread of this species, its prevalence has been reduced. Burkholderia multivorans is the now the most dominant Bcc infection encountered in the UK CF population, a changing epidemiology that also appears to be occurring in the US CF population. The distribution of Bcc species residing in the natural environment may vary considerably with the type of environment examined. Clonally identical Bcc strains have been found to occur in the natural environment and cause infection. The contamination of medical devices, disinfectants and pharmaceutical formulations has also been directly linked to several outbreaks of infection. In the last 10 years considerable progress has been made in understanding the natural biology and clinical infections caused by this fascinating group of bacteria.

A series of systematic reviews to inform a decision analysis for sampling and treating infected diabetic foot ulcers.

OBJECTIVES: To review systematically the evidence on the performance of diagnostic tests used to identify infection in diabetic foot ulcers (DFUs) and of interventions to treat infected DFUs. To use estimates derived from the systematic reviews to create a decision analytic model in order to identify the most effective method of diagnosing and treating infection and to identify areas of research that would lead to large reductions in clinical uncertainty. DATA SOURCES: Electronic databases covering period from inception of the database to November 2002. REVIEW METHODS: Selected studies were assessed against validated criteria and described in a narrative review. The structure of a decision analytic model was derived for two groups of patients in whom diagnostic tests were likely to be used. RESULTS: Three studies that investigated the performance of diagnostic tests for infection on populations including people with DFUs found that there was no evidence that single items on a clinical examination checklist were reliable in identifying infection in DFUs, that wound swabs perform poorly against wound biopsies, and that semi-quantitative analysis of wound swabs may be a useful alternative to quantitative analysis. However, few people with DFUs were included, so it was not possible to tell whether diagnostic performance differs for DFUs relative to wounds of other aetiologies. Twenty-three studies investigated the effectiveness (n = 23) or cost-effectiveness (n = 2) of antimicrobial agents for DFUs. Eight studied intravenous antibiotics, five oral antibiotics, four different topical agents such as dressings, four subcutaneous granulocyte colony stimulating factor (G-CSF), one evaluated oral and topical Ayurvedic preparations and one compared topical sugar versus antibiotics versus standard care. The majority of trials were underpowered and were too dissimilar to be pooled. There was no strong evidence for recommending any particular antimicrobial agent for the prevention of amputation, resolution of infection or ulcer healing. Topical pexiganan cream may be as effective as oral antibiotic treatment with ofloxacin for the resolution of local infection. Ampicillin and sulbactam were less costly than imipenem and cilastatin, a growth factor (G-CSF) was less costly than standard care and cadexomer iodine dressings may be less costly than daily dressings. A decision analytic model was derived for two groups of people, those for whom diagnostic testing would inform treatment--people with ulcers which do not appear infected but whose ulcer is not progressing despite optimal concurrent treatment--and those in whom a first course of antibiotics (prescribed empirically) have failed. There was insufficient information from the systematic reviews or interviews with experts to populate the model with transition probabilities for the sensitivity and specificity of diagnosis of infection in DFUs. Similarly, there was insufficient information on the probabilities of healing, amputation or death in the intervention studies for the two populations of interest. Therefore, we were unable to run the model to inform the most effective diagnostic and treatment strategy. CONCLUSIONS: The available evidence is too weak to be able to draw reliable implications for practice. This means that, in terms of diagnosis, infection in DFUs cannot be reliably identified using clinical assessment. This has implications for determining which patients need formal diagnostic testing for infection, on whether empirical treatment with antibiotics (before the results of diagnostic tests are available) leads to better outcomes, and on identifying the optimal methods of diagnostic testing. With respect to treatment, it is not known whether treatment with systemic or local antibiotics leads to better outcomes or whether any particular agent is more effective. Limited evidence suggests that both G-CSF and cadexomer iodine dressings may be less expensive than 'standard' care, that ampicillin/sulbactam may be less costly than imipenem/cilastatin, and that an unlicensed cream (pexiganan) may be as effective as oral ofloxacin. Further research is needed to ascertain the characteristics of infection in people with DFUs that influence healing and amputation outcomes, to determine whether detecting infection prior to treatment offers any benefit over empirical therapy, and to establish the most effective and cost-effective methods for detecting infection, as well as the relative effectiveness and cost-effectiveness of antimicrobial interventions for DFU infection.

Origin and molecular epidemiology of penicillin-binding-protein-mediated resistance to beta-lactam antibiotics.

Resistance to beta-lactam antibiotics in some naturally transformable bacterial pathogens has arisen by interspecies recombinational events that have generated hybrid penicillin-binding proteins with reduced affinity for the antibiotics. This type of resistance is of particular concern in pneumococci, in which it is increasing worldwide.

Data reduction in headspace analysis of blood and urine samples for robust bacterial identification.

This paper demonstrates the application of chemical headspace analysis to the problem of classifying the presence of bacteria in biomedical samples by using computational tools. Blood and urine samples of disparate forms were analysed using a Cyrano Sciences C320 electronic nose together with an Agilent 4440 Chemosensor. The high dimensional data sets resulting from these devices present computational problems for parameter estimation of discriminant models. A variety of data reduction and pattern recognition techniques were employed in an attempt to optimise the classification process. A 100% successful classification rate for the blood data from the Agilent 4440 was achieved by combining a Sammon mapping with a radial basis function neural network. In comparison a successful classification rate of 80% was achieved for the urine data from the C320 which were analysed using a novel nonlinear time series model.

Resource relationships of foraging mycelial systems of Phanerochaete velutina and Hypholoma fasciculare in soil.

Inoculum blocks of wood colonized by the basidiomycetes Hypholoma fasciculare (Huds.: Fr.) Kummer and Phanerochaete velutina (DC: Pers.) Parmasto were placed in plastic trays containing moist (matric potential -0.007 MPa), unsterilized soil. When 'baits', in the form of beech (Fagus sylvatica L.) wood blocks, descaled pine (Primus sylvestris L.) cones, beech twigs, beech leaves or pine needles were introduced, H. fasciculare consistently responded by ramification of diffuse mycelium within and over the baits, inhibition of extension and regression of non-connective mycelium, thickening of connective cords and polarized outgrowth from the baits. Decay of inoculum wood blocks was generally greater in the control than where baits were added. Whilst the decay rates of different types of baits varied, pine cones being least decayed, total loss of dry weight from the system (inocula and baits combined) was similar in all cases. The sparser, more independent and rapidly extending cords of P. velutina exhibited more varied responses to the introduction of baits, and only under certain circumstances exhibited the same responses as H. fasciculare. Although decay of inoculum blocks was generally greater in controls than where colonizable baits were added, total loss of dry weight in the latter systems was two- to fourfold greater than in controls. Snowflake-like mycelial growth patterns were induced in H. fasciculare by growing the fungus from an inoculum block placed in the centre of two concentric, hexagonal arrays of wood-block baits. Selective removal of baits from the inner hexagonal array, after they had been colonized but before renewed outgrowth from them, either greatly delayed colonization of outlying blocks, if all six were taken out, or led respectively to symmetrical or asymmetrical 'bypass' systems if three alternating or adjacent blocks were taken. These observations are further support for the interpretation of basidiomycete mycelial cords as foraging systems and suggest experimental approaches which can help to elucidate their behaviour.

Spatial dynamics and interactions of the woodland fairy ring fungus, Clitocybe nebularis.

The extension rates of Clitocybe nebularis (Batsch ex Fr.) Kummer strains on 2% malt agar were only 30-40%, of those, up to 3.4 mm d-1 , observed in woodland at equivalent exponential mean temperatures. Extension of mature field systems was accomplished by mycelial annuli or arcs 30-40 cm wide, differentiated into a leading edge of mycelial cords followed by a zone of dense, diffuse mycelium which bleached litter components, and a trailing edge of greyish, Used mycelium. Disruption of mature annuli by natural obstacles or experimental re-orientation within the mycelial band resulted in regression of the affected segment of mycelium. Localized lysis following encounter with an obstacle by immature patches of mycelium with a diameter of 30-50 cm, led to polarized development of the residual mycelium. Strains from different fruit bodies were somatically compatible when paired on 2% malt agar if sampled from the same ring, but incompatible if from different rings, resulting in mutual antagonism and formation of a persistent demarcation zone. By contrast, collision between adjacent systems in woodland culminated in mutual obliteration of the interaction fronts. C nebularis was non-combative when paired against other decomposer basidiomycetes on 2% malt agar, being either replaced or deadlocked but not replacing mycelia of these fungi. The implications of these observations are discussed in terms of emerging concepts of ecological strategies, foraging theory and polarity in mycelial collectives.

Genetics and molecular biology of beta-lactam-resistant pneumococci.

Penicillin-resistant pneumococci have been reported with increasing frequency in recent years. Isolates with high-level resistance are now found in many countries, and in some countries they constitute a substantial proportion of all isolates. A worrying development is the recent emergence of pneumococci with high-level resistance to third-generation cephalosporins. Resistance to beta-lactam antibiotics in pneumococci is due entirely to the development of altered forms of the high-molecular-weight penicillin-binding proteins (PBPs) that have decreased affinity for the antibiotics. High-level resistance to third-generation cephalosporins has occurred by the development of altered forms of PBP1a and 2x, whereas high-level penicillin resistance additionally requires alterations of PBP2b. Altered PBPs are encoded by mosaic genes that have emerged by recombinational events between the pbp genes of pneumococci and their homologs in closely related streptococcal species. Horizontal gene transfer, presumably mediated by genetic transformation, has also resulted in the dissemination of altered pbp genes, and possibly capsular biosynthetic genes, between different pneumococcal lineages to produce new resistant clones.

Molecular evolution of rifampicin resistance in Streptococcus pneumoniae.

Rifampicin resistance has arisen in several different species of bacteria because of alterations to one or more regions in the target of the antibiotic, the beta-subunit of RNA polymerase encoded by rpoB. Nucleotide sequence analysis of a 270 bp fragment of rpoB from 16 clinical rifampicin-susceptible isolates of Streptococcus pneumoniae, 8 clinical rifampicin-resistant isolates, and 3 spontaneous rifampicin-resistant mutants, has revealed that, as with previously examined species, point mutations within the cluster I region of rpoB, at sites encoding Asp516 and HiS526, also confer resistance to rifampicin in this important human pathogen. Moreover, the residues within cluster I, that were altered within the rifampicin-resistant mutants of S. pneumoniae, were in the same position as those previously found to alter in resistant isolates of Escherichia coli and Mycobacterium tuberculosis. Sequence analysis of rpoB, both from these isolates of S. pneumoniae and from two strains of S. mitis, reveals that, among a number of clinical isolates, resistance to rifampicin in S. pneumoniae has arisen by point mutation. However, the nucleotide sequence of rpoB from one isolate examined suggests that interspecies gene transfer may also have played a role in the evolution of rifampicin-resistance in S. pneumoniae.

Horizontal spread of an altered penicillin-binding protein 2B gene between Streptococcus pneumoniae and Streptococcus oralis.

The region encoding the transpeptidase domain of the penicillin-binding protein 2B (PBP 2B) gene of two penicillin-resistant clinical isolates of Streptococcus oralis was > 99.6% identical in nucleotide sequence to that of a penicillin-resistant serotype 6 isolate of Streptococcus pneumoniae. The downstream 849 base pairs of these genes were identical. Analysis of the data indicates that the PBP gene has probably been transferred from S. pneumoniae into S. oralis, rather than vice versa, and shows that one region of this resistance gene has been distributed horizontally both within S. pneumoniae and into two different viridans group streptococci.

Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae.

Mosaic penicillin-binding proteins (PBP) 1A, 2X and 2B genes were cloned from four clinical isolates of Streptococcus pneumoniae with levels of susceptibility to penicillin ranging from 1.5 to 16 micrograms benzylpenicillin ml-1. In each instance it was possible to transform either the penicillin-sensitive laboratory strain R6 or a sensitive clinical isolate 110K/70 to the full level of penicillin resistance with these three penicillin-binding proteins alone. Until now it has not been possible to clearly determine whether alterations to PBP1A, 2X and 2B alone were sufficient to attain high level penicillin resistance.

Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae.

An internal fragment from each of the penicillin-binding protein (PBP) 1A, 2B and 2X genes of Streptococcus pneumoniae, which included the region encoding the active-site serine residue, was replaced by a fragment encoding spectinomycin resistance. The resulting constructs were tested for their ability to transform S. pneumoniae strain R6 to spectinomycin resistance. Spectinomycin-resistant transformants could not be obtained using either the inactivated PBP 2X or 2B genes, suggesting that deletion of either of these genes was a lethal event, but they were readily obtained using the inactivated PBP 1A gene. Analysis using the polymerase chain reaction confirmed that the latter transformants had replaced their chromosomal copy of the PBP 1A gene with the inactivated copy of the gene. Deletion of the PBP 1A gene was therefore tolerated under laboratory conditions and appeared to have little effect on growth or susceptibility to benzylpenicillin.

ßLactam Resistance Mediated by Changes in Penicillin-Binding Proteins.

The widespread use, or perhaps overuse, of penicillin during the past 50 yr has driven the evolution of resistance to penicilling in numerous different species of bacteria.Typically, resistance has arisen as a result of the acquisition of ß-lactamases that inactivate the antibiotic (see Chapter 25 . Alternatively, in some Gram-negative bacteria, resistance may have arisen by a reduction in the ability of the antibiotic to access its target. However, in a number of clinically important Gram-negative and Gram-positive bacteria, resistance has arisen by alteration of the targets for penicillin and other ß-lactam antibiotics, namely, the penicillin-binding proteins (PBPs).