Legionella antibiotic susceptibility testing: is it time for international standardization and evidence-based guidance?

Legionella pneumophila, a Gram-negative bacillus, is the causative agent of Legionnaire's disease, a form of severe community-acquired pneumonia. Infection can have high morbidity, with a high proportion of patients requiring ICU admission, and up to 10% mortality, which is exacerbated by the lack of efficacy of typical empirical antibiotic therapy against Legionella spp. The fastidious nature of the entire Legionellaceae family historically required inclusion of activated charcoal in the solid medium to remove growth inhibitors, which inherently interferes with accurate antimicrobial susceptibility determination, an acknowledged methodological shortfall, now rectified by a new solid medium that gives results comparable to those of microbroth dilution. Here, as an international Legionella community (with authors representing various international reference laboratories, countries and clinical stakeholders for diagnosis and treatment of legionellosis), we set out recommendations for the standardization of antimicrobial susceptibility testing methods, guidelines and reference strains to facilitate an improved era of antibiotic resistance determination.

Modifiable healthcare factors affecting 28-day survival in bloodstream infection: a prospective cohort study.

BACKGROUND: Bloodstream infection is common in the UK and has significant mortality depending on the pathogen involved, site of infection and other patient factors. Healthcare staffing and ward activity may also impact on outcomes in a range of conditions, however there is little specific National Health Service (NHS) data on the impact for patients with bloodstream infection. Bloodstream Infections - Focus on Outcomes is a multicentre cohort study with the primary aim of identifying modifiable risk factors for 28-day mortality in patients with bloodstream infection due to one of six key pathogens. METHODS: Adults under the care of five NHS Trusts in England and Wales between November 2010 and May 2012 were included. Multivariable Cox regression was used to quantify the association between modifiable risk factors, including staffing levels and timing of appropriate therapy, and 28-day mortality, after adjusting for non-modifiable risk factors such as patient demographics and long-term comorbidities. RESULTS: A total of 1676 patients were included in the analysis population. Overall, 348/1676 (20.8%) died within 28 days. Modifiable factors associated with 28-day mortality were ward speciality, ward activity (admissions and discharges), movement within ward speciality, movement from critical care, and time to receipt of appropriate antimicrobial therapy in the first 7 days. For each additional admission or discharge per 10 beds, the hazard increased by 4% (95% CI 1 to 6%) in medical wards and 11% (95% CI 4 to 19%) in critical care. Patients who had moved wards within speciality or who had moved out of a critical care ward had a reduction in hazard of mortality. In the first 7 days, hazard of death increased with increasing time to receipt of appropriate antimicrobial therapy. CONCLUSION: This study underlines the importance of appropriate antimicrobials within the first 7 days, and the potential for ward activity and ward movements to impact on survival in bloodstream infection.

Towards better antimicrobial susceptibility testing: impact of the Journal of Antimicrobial Chemotherapy.

Susceptibility testing of bacteria is one of the most important tests performed in a clinical microbiology laboratory. Improvements in laboratory techniques, especially the move towards standardized susceptibility testing, has provided better consistency and accuracy of testing. When used in conjunction with the most recently developed interpretative criteria, the result is better prediction of the outcome of antimicrobial therapy for infected patients. Throughout the last four decades this Journal has published numerous articles evidencing improvements and new techniques, a valuable source of information for microbiology laboratories.

Antimicrobial susceptibility testing breakpoints and methods from BSAC to EUCAST.

The BSAC Standing Committee on Antimicrobial Susceptibility Testing is one of several European national breakpoint committees that agreed in 2002 to harmonize clinical MIC breakpoints. The process of harmonization has since been completed for commonly used agents, and breakpoints for new agents have been set by EUCAST in accordance with a procedure defined by the EMA. EUCAST breakpoints have now been adopted by a large majority of laboratories in Europe. BSAC implemented the EUCAST breakpoints in its own disc diffusion susceptibility testing method as harmonized breakpoints were agreed to over the years. Since the development of the EUCAST disc diffusion method, several countries with their own disc diffusion methods have switched to the EUCAST method, and BSAC will replace support of its own disc diffusion method with support for the EUCAST method from January 2016. The EUCAST breakpoints are also available in automated systems. The harmonized breakpoints and methods will help to avoid different reports of susceptibility for the same isolate in different countries and enable more reliable comparison of resistance rates in surveillance studies in different countries.

Non-susceptibility of Escherichia coli, Klebsiella spp., Pseudomonas spp., Streptococcus pneumoniae and Staphylococcus aureus in the UK: temporal trends in England, Northern Ireland, Scotland and Wales.

OBJECTIVES: To monitor and compare trends in the non-susceptibility of bloodstream isolates of pathogens to key antibiotics in the constituent countries of the UK between 2010 and 2014. METHODS: Routinely generated antibiotic susceptibility test results for bloodstream isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas spp., Streptococcus pneumoniae and Staphylococcus aureus were collected from hospital microbiology laboratories in each country. RESULTS: With the exception of a decrease in the proportion of S. aureus that were MRSA, non-susceptibility to key antibiotics among the pathogens studied remained largely unchanged over the 5 year study period, with any increases in non-susceptibility being small. Although some intercountry variation in the proportions of non-susceptible isolates was seen, apart from MRSA, the differences were generally small (<5%) and fluctuated from year to year, with no country showing consistently higher or lower rates of resistance. CONCLUSIONS: Collaboration between the constituent countries of the UK allows an integrated approach to nationwide surveillance of antibiotic resistance.

Improving the Diagnosis and Treatment of Urinary Tract Infection in Young Children in Primary Care: Results from the DUTY Prospective Diagnostic Cohort Study.

PURPOSE: Up to 50% of urinary tract infections (UTIs) in young children are missed in primary care. Urine culture is essential for diagnosis, but urine collection is often difficult. Our aim was to derive and internally validate a 2-step clinical rule using (1) symptoms and signs to select children for urine collection; and (2) symptoms, signs, and dipstick testing to guide antibiotic treatment. METHODS: We recruited acutely unwell children aged under 5 years from 233 primary care sites across England and Wales. Index tests were parent-reported symptoms, clinician-reported signs, urine dipstick results, and clinician opinion of UTI likelihood (clinical diagnosis before dipstick and culture). The reference standard was microbiologically confirmed UTI cultured from a clean-catch urine sample. We calculated sensitivity, specificity, and area under the receiver operator characteristic (AUROC) curve of coefficient-based (graded severity) and points-based (dichotomized) symptom/sign logistic regression models, and we then internally validated the AUROC using bootstrapping. RESULTS: Three thousand thirty-six children provided urine samples, and culture results were available for 2,740 (90%). Of these results, 60 (2.2%) were positive: the clinical diagnosis was 46.6% sensitive, with an AUROC of 0.77. Previous UTI, increasing pain/crying on passing urine, increasingly smelly urine, absence of severe cough, increasing clinician impression of severe illness, abdominal tenderness on examination, and normal findings on ear examination were associated with UTI. The validated coefficient- and points-based model AUROCs were 0.87 and 0.86, respectively, increasing to 0.90 and 0.90, respectively, by adding dipstick nitrites, leukocytes, and blood. CONCLUSIONS: A clinical rule based on symptoms and signs is superior to clinician diagnosis and performs well for identifying young children for noninvasive urine sampling. Dipstick results add further diagnostic value for empiric antibiotic treatment.

Empiric antibiotic treatment for urinary tract infection in preschool children: susceptibilities of urine sample isolates.

BACKGROUND: Antibiotic treatment recommendations based on susceptibility data from routinely submitted urine samples may be biased because of variation in sampling, laboratory procedures and inclusion of repeat samples, leading to uncertainty about empirical treatment. OBJECTIVE: To describe and compare susceptibilities of Escherichia coli cultured from routinely submitted samples, with E. coli causing urinary tract infection (UTI) from a cohort of systematically sampled, acutely unwell children. METHODS: Susceptibilities of 1458 E. coli isolates submitted during the course of routine primary care for children <5 years (routine care samples), compared to susceptibilities of 79 E. coli isolates causing UTI from 5107 children <5 years presenting to primary care with an acute illness [systematic sampling: the Diagnosis of Urinary Tract infection in Young children (DUTY) cohort]. RESULTS: The percentage of E. coli sensitive to antibiotics cultured from routinely submitted samples were as follows: amoxicillin 45.1% (95% confidence interval: 42.5-47.7%); co-amoxiclav using the lower systemic break point (BP) 86.6% (84.7-88.3%); cephalexin 95.1% (93.9-96.1%); trimethoprim 74.0% (71.7-76.2%) and nitrofurantoin 98.2% (97.4-98.8%). The percentage of E. coli sensitive to antibiotics cultured from systematically sampled DUTY urines considered to be positive for UTI were as follows: amoxicillin 50.6% (39.8-61.4%); co-amoxiclav using the systemic BP 83.5% (73.9-90.1%); co-amoxiclav using the urinary BP 94.9% (87.7-98.4%); cephalexin 98.7% (93.2-99.8%); trimethoprim 70.9% (60.1-80.0%); nitrofurantoin 100% (95.3-100.0%) and ciprofloxacin 96.2% (89.4-98.7%). CONCLUSION: Escherichia coli susceptibilities from routine and systematically obtained samples were similar. Most UTIs in preschool children remain susceptible to nitrofurantoin, co-amoxiclav and cephalexin.

Enhanced surveillance of Clostridium difficile infection occurring outside hospital, England, 2011 to 2013.

There are limited national epidemiological data for community-associated (CA)-Clostridium difficile infections (CDIs). Between March 2011 and March 2013, laboratories in England submitted to the Clostridium difficile Ribotyping Network (CDRN) up to 10 diarrhoeal faecal samples from successive patients with CA-CDI, defined here as C. difficile toxin-positive diarrhoea commencing outside hospital (or less than 48 hours after hospital admission), including those cases associated with community-based residential care, with no discharge from hospital within the previous 12 weeks. Patient demographics and C. difficile PCR ribotypes were compared for CA-CDIs in our study and presumed healthcare-associated (HA) CDIs via CDRN. Ribotype diversity indices, ranking and relative prevalences were very similar in CA- vs HA-CDIs, although ribotypes 002 (p ≤ 0.0001),020 (p = 0.009) and 056 (p < 0.0001) predominated in CA-CDIs; ribotype 027 (p = 0.01) predominated in HA-CDIs. Epidemic ribotypes 027 and 078 predominated in institutional residents with CDI (including care/nursing homes) compared with people with CDI living at home. Ribotype diversity decreased with increasing age in HA-CDIs, but not in CA-CDIs. Ribotype 078 CA-CDIs were significantly more common in elderly people (3.4% (6/174) vs 8.7% (45/519) in those aged < 65 and ≥ 65 years, respectively; p = 0.019). No antibiotics were prescribed in the previous four weeks in about twofold more CA-CDI vs HAs (38.6% (129/334) vs 20.3% (1,226/6,028); p < 0.0001). We found very similar ribotype distributions in CA- and HA-CDIs, although a few ribotypes significantly predominated in one setting. These national data emphasise the close interplay between, and likely common reservoirs for, CDIs, particularly when epidemic strains are not dominant.

Cluster of atypical adult Guillain-Barré syndrome temporally associated with neurological illness due to EV-D68 in children, South Wales, United Kingdom, October 2015 to January 2016.

We report a cluster of atypical Guillain-Barré syndrome in 10 adults temporally related to a cluster of four children with acute flaccid paralysis, over a 3-month period in South Wales, United Kingdom. All adult cases were male, aged between 24 and 77 years. Seven had prominent facial diplegia at onset. Available electrophysiological studies showed axonal involvement in five adults. Seven reported various forms of respiratory disease before onset of neurological symptoms. The ages of children ranged from one to 13 years, three of the four were two years old or younger. Enterovirus testing is available for three children; two had evidence of enterovirus D68 infection in stool or respiratory samples. We describe the clinical features, epidemiology and state of current investigations for these unusual clusters of illness.

Characterization of plasmids in extensively drug-resistant acinetobacter strains isolated in India and Pakistan.

The blaNDM-1 gene is associated with extensive drug resistance in Gram-negative bacteria. This probably spread to Enterobacteriaceae from Acinetobacter spp., and we characterized plasmids associated with blaNDM-1 in Acinetobacter spp. to gain insight into their role in this dissemination. Four clinical NDM-1-producing Acinetobacter species strains from India and Pakistan were investigated. A plasmid harboring blaNDM-1, pNDM-40-1, was characterized by whole-genome sequencing of Acinetobacter bereziniae CHI-40-1 and comparison with related plasmids. The presence of similar plasmids in strains from Pakistan was sought by PCR and sequencing of amplicons. Conjugation frequency was tested and stability of pNDM-40-1 investigated by real-time PCR of isolates passaged with and without antimicrobial selection pressure. A. bereziniae and Acinetobacter haemolyticus strains contained plasmids similar to the pNDM-BJ01-like plasmids identified in Acinetobacter spp. in China. The backbone of pNDM-40-1 was almost identical to that of pNDM-BJ01-like plasmids, but the transposon harboring blaNDM-1, Tn125, contained two short deletions. Escherichia coli and Acinetobacter pittii transconjugants were readily obtained. Transconjugants retained pNDM-40-1 after a 14-day passage experiment, although stability was greater with meropenem selection. Fragments of pNDM-BJ01-like plasmid backbones are found near blaNDM-1 in some genetic contexts from Enterobacteriaceae, suggesting that cross-genus transfer has occurred. pNDM-BJ01-like plasmids have been described in isolates originating from a wide geographical region in southern Asia. In vitro data on plasmid transfer and stability suggest that these plasmids could have contributed to the spread of blaNDM-1 into Enterobacteriaceae.

A demonstration of the susceptibility of clinical isolates obtained from cystic fibrosis patients to manuka honey.

Pseudomonas and Burkholderia pose a significant health threat to people with chronic respiratory conditions; the resistance inherent in these bacteria indicates that new antimicrobial strategies are required. Susceptibility of 56 strains of P. aeruginosa and 55 strains of Burkholderia to manuka honey, tobramycin and colistin using microbroth dilution and E strip was determined. MICs of antibiotics with honey were determined to search for synergistic combinations against two representative strains of each genus. All strains exhibited susceptibility to honey ≤10 % (w/v); mean susceptibility of Burkholderia (4.6 % w/v) was lower than P. aeruginosa (7.3 % w/v). Synergistic or additive combinations were found with all four strains tested. Combinations of manuka honey with antibiotics can be used to lower the MIC need to successfully inhibit both P. aeruginosa and B. cepacia. The use of honey as a combination agent may be possible for the management of P. aeruginosa and B. cepacia.

Antibiotic prescribing and associated diarrhoea: a prospective cohort study of care home residents.

BACKGROUND: the risk factors for and frequency of antibiotic prescription and antibiotic-associated diarrhoea (AAD) among care home residents are unknown. AIM: to prospectively study frequency and risks for antibiotic prescribing and AAD for care home residents. DESIGN AND SETTING: a 12-month prospective cohort study in care homes across South Wales. METHOD: antibiotic prescriptions and the development of AAD were recorded on case report forms. We defined AAD as three or more loose stools in a 24-h period occurring within 8 weeks of exposure to an antibiotic. RESULTS: we recruited 279 residents from 10 care homes. The incidence of antibiotic prescriptions was 2.16 prescriptions per resident year (95% CI: 1.90-2.46). Antibiotics were less likely to be prescribed to residents from dual-registered homes (OR compared with nursing homes: 0.38, 95% CI: 0.18-0.79). For those who were prescribed antibiotics, the incidence of AAD was 0.57 episodes per resident year (95% CI: 0.41-0.81 episodes). AAD was more likely in residents who were prescribed co-amoxiclav (hazards ratio, HR = 2.08, 95% confidence interval, CI: 1.18-3.66) or routinely used incontinence pads (HR = 2.54, 95% CI: 1.26-5.13) and less likely in residents from residential homes (HR compared with nursing homes: 0.14, 95% CI: 0.06-0.32). CONCLUSION: residents of care homes, particularly of nursing homes, are frequently prescribed antibiotics and often experience diarrhoea following such prescriptions. Co-amoxiclav is associated with greater risk of AAD.

Plasmid carriage of bla NDM-1 in clinical Acinetobacter baumannii isolates from India.

NDM-1 probably emerged in Acinetobacter species prior to its dissemination among Enterobacteriaceae, and NDM-1-like enzymes are increasingly reported in Acinetobacter species. Here, we report on the genetic context of blaNDM-1 in the earliest known NDM-1-producing organisms, clinical isolates of Acinetobacter from India in 2005. These strains harbor blaNDM-1 plasmids of different sizes. The gene is associated with the remnants of the Tn125 transposon normally associated with blaNDM-1 in Acinetobacter spp. The transposon has been disrupted by the IS26 insertion and subsequent movement events.

Point of care testing for urinary tract infection in primary care (POETIC): protocol for a randomised controlled trial of the clinical and cost effectiveness of FLEXICULT™ informed management of uncomplicated UTI in primary care.

BACKGROUND: Urinary tract infections (UTI) are the most frequent bacterial infection affecting women and account for about 15% of antibiotics prescribed in primary care. However, some women with a UTI are not prescribed antibiotics or are prescribed the wrong antibiotics, while many women who do not have a microbiologically confirmed UTI are prescribed antibiotics. Inappropriate antibiotic prescribing unnecessarily increases the risk of side effects and the development of antibiotic resistance, and wastes resources. METHODS/DESIGN: 614 adult female patients will be recruited from four primary care research networks (Wales, England, Spain, the Netherlands) and individually randomised to either POCT guided care or the guideline-informed 'standard care' arm. Urine and stool samples (where possible) will be obtained at presentation (day 1) and two weeks later for microbiological analysis. All participants will be followed up on the course of their illness and their quality of life, using a 2 week self-completed symptom diary. At 3 months, a primary care notes review will be conducted for evidence of further evidence of treatment failures, recurrence, complications, hospitalisations and health service costs. DISCUSSION: Although the Flexicult™ POCT is used in some countries in routine primary care, it's clinical and cost effectiveness has never been evaluated in a randomised clinical trial. If shown to be effective, the use of this POCT could benefit individual sufferers and provide evidence for health care authorities to develop evidence based policies to combat the spread and impact of the unprecedented rise of infections caused by antibiotic resistant bacteria in Europe. TRIAL REGISTRATION NUMBER: ISRCTN65200697 (Registered 10 September 2013).

Genomic epidemiology reveals geographical clustering of multidrug-resistant Escherichia coli ST131 associated with bacteraemia in Wales.

Antibiotic resistance is a significant global public health concern. Uropathogenic Escherichia coli sequence type (ST)131, a widely prevalent multidrug-resistant clone, is frequently associated with bacteraemia. This study investigates third-generation cephalosporin resistance in bloodstream infections caused by E. coli ST131. From 2013-2014 blood culture surveillance in Wales, 142 E. coli ST131 genomes were studied alongside global data. All three major ST131 clades were represented across Wales, with clade C/H30 predominant (n = 102/142, 71.8%). Consistent with global findings, Welsh strains of clade C/H30 contain ß-lactamase genes from the blaCTX-M-1 group (n = 65/102, 63.7%), which confer resistance to third-generation cephalosporins. Most Welsh clade C/H30 genomes belonged to sub-clade C2/H30Rx (58.3%). A Wales-specific sub-lineage, named GB-WLS.C2, diverged around 1996-2000. An introduction to North Wales around 2002 led to a localised cluster by 2009, depicting limited genomic diversity within North Wales. This investigation emphasises the value of genomic epidemiology, allowing the detection of genetically similar strains in local areas, enabling targeted and timely public health interventions.

Test performance of lateral flow rapid antigen tests for COVID-19 in Welsh adult care home staff using routine surveillance data.

BACKGROUND: Lateral flow tests (LFTs) have been used to screen for SARS-CoV2 in Wales since January 2021. Between May and August 2021, adult care home staff policy was for biweekly Innova LFT and weekly Polymerase Chain Reaction (PCR) testing while asymptomatic. We estimated test performance of LFTs conducted in adult care home staff using PCR tests as a reference standard. METHODS: Test results from surveillance data were matched by individual where both LFT and PCR were taken on the same day. We calculated sensitivity, specificity, positive and negative predictive values, and agreement using Matthew's correlation coefficient. Ct values of positive PCR results were compared by matched LFT result. Analysis was conducted using R v4.1.3. RESULTS: We analysed 115,593 test pairs, 499 (0.43%) of which were PCR positive. Median age was 48 (IQR: 22) and 85.00% of the study population were female. Test result agreement was 99.59% (95%CI 99.55-99.63; MCC: 0.38, p<0.001). Sensitivity and specificity were 25.65% (95%CI 22.02-29.67) and 99.91% (95%CI 99.89-99.93), respectively. PPV was 55.90% (95%CI 49.42-62.17) and NPV was 99.68% (95%CI 99.64-99.71). Crude Ct values were significantly lower in positive PCR tests matched to a positive LFT compared to a negative LFT. CONCLUSIONS: Specificity and negative predictive value were high in an asymptomatic population of care home staff indicating this test is an effective tool for identifying cases of SARS-CoV-2 infection during periods of high prevalence where transmission is likely, due to the presence of high viral loads. Positive predictive value results are lower than existing literature yet should be considered in light of the asymptomatic study population and low prevalence (under 1%) at the time most of these tests were conducted. Performance improved at times of higher prevalence during the study. These results suggest that whilst lateral flow tests are effective for identifying SARS-COV-2 infections with high viral loads, they are not effective at identifying cases with a low viral load. When an LFT provides a negative result, false negatives should be considered and additional diagnostic tests performed.

Overcoming drug resistance with alginate oligosaccharides able to potentiate the action of selected antibiotics.

The uncontrolled, often inappropriate use of antibiotics has resulted in the increasing prevalence of antibiotic-resistant pathogens, with major cost implications for both United States and European health care systems. We describe the utilization of a low-molecular-weight oligosaccharide nanomedicine (OligoG), based on the biopolymer alginate, which is able to perturb multidrug-resistant (MDR) bacteria by modulating biofilm formation and persistence and reducing resistance to antibiotic treatment, as evident using conventional and robotic MIC screening and microscopic analyses of biofilm structure. OligoG increased (up to 512-fold) the efficacy of conventional antibiotics against important MDR pathogens, including Pseudomonas, Acinetobacter, and Burkholderia spp., appearing to be effective with several classes of antibiotic (i.e., macrolides, ß-lactams, and tetracyclines). Using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), increasing concentrations (2%, 6%, and 10%) of alginate oligomer were shown to have a direct effect on the quality of the biofilms produced and on the health of the cells within that biofilm. Biofilm growth was visibly weakened in the presence of 10% OligoG, as seen by decreased biomass and increased intercellular spaces, with the bacterial cells themselves becoming distorted and uneven due to apparently damaged cell membranes. This report demonstrates the feasibility of reducing the tolerance of wound biofilms to antibiotics with the use of specific alginate preparations.