Evaluation of the Haemophilus influenzae EUCAST and CLSI disc diffusion methods to recognize aminopenicillin and amoxicillin/clavulanate resistance.

OBJECTIVES: Implementation of EUCAST susceptibility testing in an Australian hospital laboratory demonstrated higher rates of aminopenicillin and amoxicillin/clavulanate resistance in Haemophilus influenzae than previously recognized. This study aimed to better define the variability in the detection of ß-lactam resistance based on EUCAST and CLSI disc diffusion (DD) methodology, by comparison with the recommended reference method, broth microdilution (BMD), and by concordance with genomic analysis. METHODS: A total of 100 random H. influenzae isolates were assessed for ampicillin and amoxicillin/clavulanate susceptibility by EUCAST and CLSI DD and BMD. WGS was used to analyse the ftsI gene of a subset of isolates with ß-lactam resistance, other than that due to isolated ß-lactamase production. RESULTS: Of the 100 isolates, 32 were categorized as either ß-lactamase negative, ampicillin resistant (BLNAR) (n = 18) or ß-lactamase positive, amoxicillin/clavulanate resistant (BLPACR) (n = 14) by EUCAST DD. All 18 EUCAST BLNAR isolates were genotypically confirmed by WGS. Five of 18 BLNAR isolates were concordant by CLSI DD, 12 by EUCAST BMD and 4 by CLSI BMD. Nine of 14 EUCAST BLPACR isolates were confirmed by WGS; the remaining 5 were 1 mm below the EUCAST DD breakpoint. Only one isolate was detected as BLPACR by CLSI DD. Group III mutations associated with high-level ampicillin resistance were identified in 10/32 isolates. CONCLUSIONS: The EUCAST DD susceptibility method is more reliable than either CLSI or BMD for the detection of genotypically defined BLNAR resistance. However, accurate categorization of amoxicillin/clavulanate resistance remains problematic. Continuous and reproducible surveillance of resistance is needed; for this to be possible, robust susceptibility methods are required.

Genomic dynamics of species and mobile genetic elements in a prolonged blaIMP-4-associated carbapenemase outbreak in an Australian hospital.

BACKGROUND: Hospital outbreaks of carbapenemase-producing organisms, such as blaIMP-4-containing organisms, are an increasing threat to patient safety. OBJECTIVES: To investigate the genomic dynamics of a 10 year (2006-15) outbreak of blaIMP-4-containing organisms in a burns unit in a hospital in Sydney, Australia. METHODS: All carbapenem-non-susceptible or MDR clinical isolates (2006-15) and a random selection of equivalent or ESBL-producing environmental isolates (2012-15) were sequenced [short-read (Illumina), long-read (Oxford Nanopore Technology)]. Sequence data were used to assess genetic relatedness of isolates (Mash; mapping and recombination-adjusted phylogenies), perform in silico typing (MLST, resistance genes and plasmid replicons) and reconstruct a subset of blaIMP plasmids for comparative plasmid genomics. RESULTS: A total of 46/58 clinical and 67/96 environmental isolates contained blaIMP-4. All blaIMP-4-positive organisms contained five or more other resistance genes. Enterobacter cloacae was the predominant organism, with 12 other species mainly found in either the environment or patients, some persisting despite several cleaning methods. On phylogenetic analysis there were three genetic clusters of E. cloacae containing both clinical and environmental isolates, and an additional four clusters restricted to either reservoir. blaIMP-4 was mostly found as part of a cassette array (blaIMP-4-qacG2-aacA4-catB3) in a class 1 integron within a previously described IncM2 plasmid (pEl1573), with almost complete conservation of this cassette across the species over the 10 years. Several other plasmids were also implicated, including an IncF plasmid backbone not previously widely described in association with blaIMP-4. CONCLUSIONS: Genetic backgrounds disseminating blaIMP-4 can persist, diversify and evolve amongst both human and environmental reservoirs during a prolonged outbreak despite intensive prevention efforts.

Microsphaeropsis arundinis skin and soft tissue infection in renal transplant recipients: three case reports and a review of the literature.

Microsphaeropsis arundinis, a dematiaceous mold, is emerging as a cause of skin and soft tissue infection in immunocompromised hosts. Diagnosis is challenging because of the difficulty in identifying Microsphaeropsis species morphologically and few data are available to guide optimal management. We report 3 renal transplant recipients with M. arundinis soft tissue infection, where the etiological agent was diagnosed using DNA sequencing, and who were successfully treated with prolonged courses of extended-spectrum triazole antifungal agents.

Consensus guidelines for the treatment of invasive mould infections in haematological malignancy and haemopoietic stem cell transplantation, 2014.

Mould species represent the pathogens most commonly associated with invasive fungal disease in patients with haematological malignancies and patients of haemopoietic stem cell transplants. Invasive mould infections in these patient populations, particularly in the setting of neutropenia, are associated with high morbidity and mortality, and significantly increase the complexity of management. While Aspergillus species remain the most prevalent cause of invasive mould infections, Scedosporium and Fusarium species and the Mucormycetes continue to place a significant burden on the immunocompromised host. Evidence also suggests that infections caused by rare and emerging pathogens are increasing within the setting of broad-spectrum antifungal prophylaxis and improved survival times placing immunosuppressed patients at risk for longer. These guidelines present evidence-based recommendations for the antifungal management of common, rare and emerging mould infections in both adult and paediatric populations. Where relevant, the role of surgery, adjunctive therapy and immunotherapy is also discussed.

Consensus guidelines for the use of empiric and diagnostic-driven antifungal treatment strategies in haematological malignancy, 2014.

Invasive fungal disease (IFD) causes significant morbidity and mortality in patients undergoing allogeneic haemopoietic stem cell transplantation or chemotherapy for haematological malignancy. Much of these adverse outcomes are due to the limited ability of traditional diagnostic tests (i.e. culture and histology) to make an early and accurate diagnosis. As persistent or recurrent fevers of unknown origin (PFUO) in neutropenic patients despite broad-spectrum antibiotics have been associated with the development of IFD, most centres have traditionally administered empiric antifungal therapy (EAFT) to patients with PFUO. However, use of an EAFT strategy has not been shown to have an overall survival benefit and is associated with excessive antifungal therapy use. As a result, the focus has shifted to developing more sensitive and specific diagnostic tests for early and more targeted antifungal treatment. These tests, including the galactomannan enzyme-linked immunosorbent assay and Aspergillus polymerase chain reaction (PCR), have enabled the development of diagnostic-driven antifungal treatment (DDAT) strategies, which have been shown to be safe and feasible, reducing antifungal usage. In addition, the development of effective antifungal prophylactic strategies has changed the landscape in terms of the incidence and types of IFD that clinicians have encountered. In this review, we examine the current role of EAFT and provide up-to-date data on the newer diagnostic tests and algorithms available for use in EAFT and DDAT strategies, within the context of patient risk and type of antifungal prophylaxis used.

Optimal phenotypic testing of AmpC beta-lactamases using boronic acid solutions.

Plasmid-mediated class C ß-lactamases are reported from Escherichia coli and Klebsiella pneumoniae with increasing frequency. No screening and confirmatory tests have been uniformly established for these strains. We investigated for the presence of plasmid-mediated AmpC production in 51 clinical isolates of Enterobacteriaceae, comparing two different boronic acid formulations, phenylboronic acid (PB) and 3-(N-Boc-amino)phenylboronic acid (APB), using polymerase chain reaction (PCR). PB performed better than APB.

Persistent Rhodococcus equi infection in a renal transplant patient: case report and review of the literature.

Rhodococcus equi is a pathogen that mainly causes infection in immunocompromised hosts. We report a case of relapsing R. equi pulmonary infection in a 57-year-old male renal transplant recipient who was treated with 12 months of antibiotics, adjunctive surgery, and a reduction in his immunosuppression. He suffered from relapsing disease, treatment-related complications, and ultimately died of Pneumocystis pneumonia. Case reports in the literature portray a good cure rate for transplant-related R. equi infections, with shorter durations of antibiotics. The case illustrates the difficulties in the management of R. equi infections. Forty cases from the literature were reviewed and compared in terms of epidemiology, location of infection, transplant type, immunosuppression used, treatment used, outcomes, and possible exposures.

Sickness presenteeism associated with influenza-like illness in veterinarians working in New South Wales: Results of a state-wide survey.

BACKGROUND: Sickness presenteeism in the veterinary profession potentially jeopardises the wellbeing of veterinary team members and endangers quality of patient care. In veterinary team members with influenza-like illness (ILI), sickness presenteeism poses a risk to the health and wellbeing of colleagues and clients, particularly in the context of the COVID-19 pandemic. This study aimed to evaluate factors associated with sickness presenteeism in NSW registered veterinarians suffering from ILI, both before and since the beginning of the COVID-19 pandemic. METHODS: Veterinarians registered in NSW were invited to complete an anonymous online mixed-methods survey between 31 March 2021 and 31 June 2021, regarding sickness presenteeism and absenteeism associated with ILI. The questionnaire was distributed through online and print newsletters of the Australian Veterinary Association NSW Branch and the NSW Veterinary Practitioners Board. RESULTS: From a total of 122 participants, 81 veterinarians (66.4%) reported that they would attend work despite displaying symptoms of ILI. Most veterinarians would stay at home with a fever alone (n = 108, 88.5%), however, many would still attend work with a sore throat (n = 121, 99.2%) or a dry cough (n = 91, 74.6%). Sickness presenteeism was significantly associated with lack of staff to cover workers. Although sickness presenteeism remained common, participants reported that they were less likely to attend work with symptoms of ILI since the beginning of the COVID-19 pandemic. DISCUSSION: The data are discussed in relation to sickness presenteeism in healthcare workers. These findings underscore an urgent need for relief staff to decrease sickness presenteeism.

Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells.

Treatment with cytochalasin D, a drug that acts by inducing the depolymerization of the actin cytoskeleton, selectively blocked endocytosis of membrane bound and fluid phase markers from the apical surface of polarized MDCK cells without affecting the uptake from the basolateral surface. Thus, in MDCK cell transformants that express the VSV G protein, cytochalasin blocked the internalization of an anti-G mAb bound to apical G molecules, but did not reduce the uptake of antibody bound to the basolateral surface. The selective effect of cytochalasin D on apical endocytosis was also demonstrated by the failure of the drug to reduce the uptake of 125I-labeled transferrin, which occurs by receptor-mediated endocytosis, via clathrin-coated pits, almost exclusively from the basolateral surface. The actin cytoskeleton appears to play a critical role in adsorptive as well as fluid phase apical endocytic events, since treatment with cytochalasin D prevented the apical uptake of cationized ferritin, that occurs after the marker binds to the cell surface, as well as uptake of Lucifer yellow, a fluorescent soluble dye. Moreover, the drug efficiently blocked infection of the cells with influenza virus, when the viral inoculum was applied to the apical surface. On the other hand, it did not inhibit the basolateral uptake of Lucifer yellow, nor did it prevent infection with VSV from the basolateral surface, or with influenza when this virus was applied to monolayers in which the formation of tight junctions had been prevented by depletion of calcium ions. EM demonstrated that cytochalasin D leads to an increase in the number of coated pits in the apical surface where it suppresses the pinching off of coated vesicles. In addition, in drug-treated cells cationized ferritin molecules that were bound to microvilli were not cleared from the microvillar surface, as is observed in untreated cells. These findings indicate that there is a fundamental difference in the process by which endocytic vesicles are formed at the two surfaces of polarized epithelial cells and that the integrity and/or the polymerization of actin filaments are required at the apical surface. Actin filaments in microvilli may be part of a mechanochemical motor that moves membrane components along the microvillar surface towards intermicrovillar spaces, or provides the force required for converting a membrane invagination or pit into an endocytic vesicle within the cytoplasm.

Sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells.

Previous studies (Rindler, M. J., I. E., Ivanov, H. Plesken, and D. D. Sabatini, 1985, J. Cell Biol., 100: 136-151; Rindler, M. J., I. E. Ivanov, H. Plesken, E. J. Rodriguez-Boulan, and D. D. Sabatini, 1984, J. Cell Biol., 98: 1304-1319) have demonstrated that in polarized Madin-Darby canine kidney cells infected with vesicular stomatitis virus (VSV) or influenza virus the viral envelope glycoproteins G and HA are segregated to the basolateral and apical plasma membrane domains, respectively, where budding of the corresponding viruses takes place. Furthermore, it has been shown that this segregation of the glycoproteins reflects the polarized delivery of the newly synthesized polypeptides to each surface domain. In transfection experiments using eukaryotic expression plasmids that contain cDNAs encoding the viral glycoproteins, it is now shown that even in the absence of other viral components, both proteins are effectively segregated to the appropriate cell surface domain. In transfected cells, the HA glycoprotein was almost exclusively localized in the apical cell surface, whereas the G protein, although preferentially localized in the basolateral domains, was also present in lower amounts, in the apical surfaces of many cells. Using transfected and infected cells, it was demonstrated that, after reaching the cell surface, the G protein, but not the HA protein, undergoes interiorization by endocytosis. Thus, in the presence of chloroquine, a drug that blocks return of interiorized plasma membrane proteins to the cell surface, the G protein was quantitatively trapped in endosome- or lysosome-like vesicles. The sequestration of G was a rapid process that was completed in many cells by 1-2 h after chloroquine treatment. The fact that in transfected cells the surface content of G protein was not noticeably reduced during a 5-h incubation with cycloheximide, a protein synthesis inhibitor that did not prevent the effect of chloroquine, implies that normally, G protein molecules are not only interiorized but are also recycled to the cell surface.

Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination.

The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cells, is defective in DNA double-strand break repair and in ability to undergo V(D)J recombination. The human XRCC5 DNA repair gene, which complements this mutant, is shown here through genetic and biochemical evidence to be the 80-kilodalton subunit of the Ku protein. Ku binds to free double-stranded DNA ends and is the DNA-binding component of the DNA-dependent protein kinase. Thus, the Ku protein is involved in DNA repair and in V(D)J recombination, and these results may also indicate a role for the Ku-DNA-dependent protein kinase complex in those same processes.

A survey of medical staff attitudes to an antibiotic approval and stewardship programme.

BACKGROUND: Antibiotic stewardship programmes (ASPs) are advocated to ensure appropriate antimicrobial use; with short-term evidence they may improve outcomes, limit adverse effects, encourage cost-effectiveness and reduce antibiotic resistance. At Concord Hospital, a 450-bed acute care hospital, we have used a telephone-based ASP for 15 years. There may be differences in attitudes to the ASP by prescribers, which may influence its long-term efficacy. METHOD: A 40-item self-administered questionnaire was sent to 190 junior and 250 specialist medical staff. We aimed to elicit medical staff attitudes to the ASP's utility, educational value, effect on patient care and ease of use. RESULTS: One hundred and sixty-four completed questionnaires were returned. Most (82%, 95% confidence interval (95%CI) 75-87%) clinicians had used the ASP, 98% of whom believed it to be a reasonable system. Most staff (85%, 95%CI 79-90%) believed that seeking approval made teams think carefully about antibiotic choice, agreed it provided helpful advice (91%, 95%CI 85-95%) and that the approval system provided useful advice and was educational (88%, 95%CI 81-92%). The ASP was felt time-consuming and detracting from clinical duties by 33% (95%CI 26-41%), while 10% (95%CI 5.8-15.7%) believed it undervalued intuition and experience. Few (19%, 95%CI 13-25%, P < 0.0001) clinicians believed it infringed their autonomy. The advice given through the ASP was believed by most (89%, 95%CI 81-92%) to improve patient outcomes. CONCLUSION: The ASP was surprisingly well supported by all levels of staff, and reinforced the benefits of maintaining an ASP policy.

Protein kinases and DNA damage.

Failure to remove DNA damage is potentially lethal. Eukaryotic cells have therefore devised highly effective ways of detecting and repairing DNA lesions. Recent evidence indicates that protein kinases play essential roles in recognizing DNA damage and in transducing DNA damage signals to bring about changes in cellular metabolism that facilitate DNA repair.

Increased detection of carbapenemase-producing Enterobacteriaceae on post-clean sampling of a burns unit's wet surfaces.

Wet surface biofilms are a potential reservoir for multidrug-resistant Gram-negative organisms, including carbapenemase-producing Enterobacteriaceae (CPE). Recognition of environmental sources is important in reducing secondary patient transmission. We report the increased detection of blaIMP-4+ CPE in environmental samples from floor drains in burns unit shower rooms, when collected following cleaning as compared to pre-cleaning. We propose that disruption of biofilms during cleaning may account for the increased detection of multi-resistant organisms. The results highlight the role of the wet environment as an under-recognized potential source of CPE transmission. Environmental screening focusing on pre-cleaning samples alone will likely underestimate environmental contamination.

Point-of-care urinary pneumococcal antigen test in the emergency department for community acquired pneumonia.

BACKGROUND: Streptococcus pneumoniae is the most common cause of community-acquired pneumonia (CAP). Early diagnosis would allow more directed therapy and confidence in appropriate treatment for a majority of patients. The BinaxNOW pneumococcal urinary antigen (PNAG) test has been evaluated at laboratory level and is easy to perform and interpret, but its use as a point-of-care test has not been evaluated. A study was undertaken to assess whether PNAG testing can be reliably performed and interpreted by staff in an adult emergency department and whether rapid results influence initial treatment decisions. METHODS: Community-living adult patients presenting to the emergency department with clinical and radiological findings of pneumonia had PNAG testing performed on the same sample in both the emergency department and the microbiology laboratory in a blinded fashion. Accuracy and turnaround time were assessed. Diagnostic yield was compared with routine culture methods. RESULTS: Fifty-nine patients were enrolled of whom nine (15%) had positive PNAG tests. These included three culture-proven cases and six additional cases. There was 98% concordance between emergency department and laboratory results. Turnaround time was significantly shorter when tested in the emergency department (median 2 h 39 min vs 19 h 40 min). Antibiotic prescribing was not influenced by results in this small sample. CONCLUSIONS: PNAG diagnosis of pneumococcal pneumonia can be accurately performed as a point-of-care test by emergency department clinical staff. Without specific efforts to achieve early urine collection, the timeframe of testing will frequently fall outside the 4-hour patient stay of a UK emergency department and may be more appropriately considered as a test for the medical admissions unit in this setting. Sensitivity is at least equal to conventional culture methods and the result is available rapidly enough to potentially influence treatment decisions, a strategy that warrants further investigation.

An international survey of cleaning and disinfection practices in the healthcare environment.

BACKGROUND: Antimicrobial resistance has become an urgent global health priority. Basic hygiene practices and cleaning and disinfection of the hospital environment are key in preventing pathogen cross-transmission. AIM: To our knowledge no studies have assessed the worldwide differences in cleaning and disinfection practices in healthcare facilities. The electronic survey described here was developed in order to evaluate differences in healthcare facility cleaning practices around the world. METHODS: The International Society of Antimicrobial Chemotherapy (ISAC, formerly ISC), Infection Prevention and Control work group developed a survey with 30 multiple-choice questions. The questions were designed to assess the current cleaning practices in healthcare settings around the world. FINDINGS: A total of 110 healthcare professionals, representing 23 countries, participated in the online survey. In 96% of the facilities a written cleaning policy was present. Training of cleaning staff occurred in 70% of the facilities at the start of employment. Cleaning practices and monitoring of these practices varied. CONCLUSIONS: The survey enabled assessment and recognition of widely differing global practices in approaches to environmental cleaning and disinfection. Development of guideline recommendations for cleaning and disinfection could improve practices and set minimum standards worldwide.

Cell cycle regulation of RNA polymerase III transcription.

Inactivation of the TATA-binding protein-containing complex TFIIIB contributes to the mitotic repression of RNA polymerase III transcription, both in frogs and in humans (J. M. Gottesfeld, V. J. Wolf, T. Dang, D. J. Forbes, and P. Hartl, Science 263:81-84, 1994; R. J. White, T. M. Gottlieb, C. S. Downes, and S. P. Jackson, Mol. Cell. Biol. 15:1983-1992, 1995). Using extracts of synchronized proliferating HeLa cells, we show that TFIIIB activity remains low during the early part of G1 phase and increases only gradually as cells approach S phase. As a result, the transcription of all class III genes tested is significantly less active in early G1 than it is in S or G2 phase, both in vitro and in vivo. The increased activity of TFIIIB as cells progress through interphase appears to be due to changes in the TATA-binding protein-associated components of this complex. The data suggest that TFIIIB is an important target for the cell cycle regulation of RNA polymerase III transcription during both mitosis and interphase of actively proliferating HeLa cells.

Mitotic regulation of a TATA-binding-protein-containing complex.

The mitotic state is associated with a generalized repression of transcription. We show that mitotic repression of RNA polymerase III transcription can be reproduced by using extracts of synchronized HeLa cells. We have used this system to investigate the molecular basis of transcriptional repression during mitosis. We find a specific decrease in the activity of the TATA-binding-protein (TBP)-containing complex TFIIIB. TBP itself is hyperphosphorylated at mitosis, but this does not appear to account for the loss of TFIIIB activity. Instead, one or more TBP-associated components appear to be regulated. The data suggest that changes in the activity of TBP-associated components contribute to the coordinate repression of gene expression that occurs at mitosis.

Future trends in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection: An in-depth review of newer antibiotics active against an enduring pathogen.

Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a major public health problem. Vancomycin and teicoplanin have been in clinical use for several decades but their drawbacks are well described. In the last 10 years, several antibiotics have been made available for clinical use. Daptomycin and linezolid have been extensively used during this period. Other agents such as ceftaroline, ceftobiprole, dalbavancin, oritavancin, tedizolid and telavancin have been approved by regulatory agencies since 2009. Many others, such as the newer tetracyclines, fluoroquinolones, oxazolidinones and pleuromutilins, are in various stages of development. In addition, an ongoing multicentre trial is investigating the role of combination of vancomycin or daptomycin with ß-lactam antibiotics. This review discusses the role of the newer antibiotics, reflecting the views of the 6th MRSA Consensus Conference meeting of the International Society of Chemotherapy MRSA Working Group that took place in 2016.

Clinical efficacy of ß-lactam/ß-lactamase inhibitor combinations for the treatment of bloodstream infection due to extended-spectrum ß-lactamase-producing Enterobacteriaceae in haematological patients with neutropaenia: a study protocol for a retrospective observational study (BICAR).

INTRODUCTION: Bloodstream infection (BSI) due to extended-spectrum ß-lactamase-producing Gram-negative bacilli (ESBL-GNB) is increasing at an alarming pace worldwide. Although ß-lactam/ß-lactamase inhibitor (BLBLI) combinations have been suggested as an alternative to carbapenems for the treatment of BSI due to these resistant organisms in the general population, their usefulness for the treatment of BSI due to ESBL-GNB in haematological patients with neutropaenia is yet to be elucidated. The aim of the BICAR study is to compare the efficacy of BLBLI combinations with that of carbapenems for the treatment of BSI due to an ESBL-GNB in this population. METHODS AND ANALYSIS: A multinational, multicentre, observational retrospective study. Episodes of BSI due to ESBL-GNB occurring in haematological patients and haematopoietic stem cell transplant recipients with neutropaenia from 1 January 2006 to 31 March 2015 will be analysed. The primary end point will be case-fatality rate within 30 days of onset of BSI. The secondary end points will be 7-day and 14-day case-fatality rates, microbiological failure, colonisation/infection by resistant bacteria, superinfection, intensive care unit admission and development of adverse events. SAMPLE SIZE: The number of expected episodes of BSI due to ESBL-GNB in the participant centres will be 260 with a ratio of control to experimental participants of 2. ETHICS AND DISSEMINATION: The protocol of the study was approved at the first site by the Research Ethics Committee (REC) of Hospital Universitari de Bellvitge. Approval will be also sought from all relevant RECs. Any formal presentation or publication of data from this study will be considered as a joint publication by the participating investigators and will follow the recommendations of the International Committee of Medical Journal Editors (ICMJE). The study has been endorsed by the European Study Group for Bloodstream Infection and Sepsis (ESGBIS) and the European Study Group for Infections in Compromised Hosts (ESGICH).