Parallel evolution of linezolid-resistant Staphylococcus aureus in patients with cystic fibrosis.

Linezolid is an antibiotic used to treat serious Staphylococcus aureus infections. Resistance to linezolid is considered rare but could emerge with repeated dosing. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). The goals of this study were to determine the incidence of linezolid-resistant methicillin-resistant Staphylococcus aureus (MRSA) in CF and identify molecular mechanisms for linezolid resistance. We identified patients who cultured S. aureus resistant to linezolid with minimum inhibitory concentration (MIC) >4 at the University of Iowa CF Center between 2008 and 2018. We obtained isolates from these patients and retested susceptibility to linezolid using broth microdilution. We used whole genome sequencing to perform phylogenetic analysis of linezolid-resistant isolates and examine sequences for mutations or accessory genes that confer linezolid resistance. Between 2008 and 2018, 111 patients received linezolid, and 4 of these patients cultured linezolid-resistant S. aureus. We sequenced 11 resistant and 21 susceptible isolates from these 4 subjects. Phylogenetic analysis indicated that linezolid resistance developed in ST5 or ST105 backgrounds. Three individuals had linezolid-resistant S. aureus with a G2576T mutation in 23S rRNA. One of these subjects additionally had a mutS- mutL- hypermutating S. aureus that produced five resistant isolates with multiple ribosomal subunit mutations. In one subject, the genetic basis for linezolid resistance was unclear. We conclude that linezolid resistant S. aureus can occur through multiple genetic mechanisms in patients with repeated exposure to this antibiotic. IMPORTANCE Patients with cystic fibrosis have persistent lung infections with Staphylococcus aureus that require extensive antibiotic treatments. Linezolid, an antibiotic given by oral or intravenous route, is prescribed repeatedly for patients whose lung disease has progressed. After treatment with linezolid, S. aureus strains can evolve antibiotic resistance through multiple genetic mechanisms. In addition to a common mutation in the 23S ribosomal RNA known to confer linezolid resistance, S. aureus strains can evolve novel resistance based on a combination of mutations affecting the bacterial ribosome. This combination of mutations was observed in a strain that exhibited hypermutation owing to the loss of the DNA repair genes mutS and mutL. In this cohort of patients with cystic fibrosis, linezolid resistance was transient, possibly due to the growth disadvantage of resistant strains. However, ongoing chronic exposure to linezolid may create optimal conditions for the future emergence of resistance to this critical antibiotic.

Parallel Evolution of Linezolid Resistant Staphylococcus aureus in Patients with Cystic Fibrosis.

Background: Linezolid is an antibiotic used to treat serious Staphylococcus aureus infections. Resistance to linezolid is considered rare but could emerge with repeated dosing. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). Objectives: The goals of this study were to determine the incidence of linezolid resistance in CF and identify molecular mechanisms for linezolid resistance. Methods: We identified patients with S. aureus resistant to linezolid (MIC > 4) at the University of Iowa CF Center between 2008 and 2018. We obtained isolates from these patients and retested susceptibility to linezolid using broth microdilution. We used whole genome sequencing to perform phylogenetic analysis of linezolid resistant isolates and examine sequences for mutations or accessory genes that confer linezolid resistance. Main Results: Between 2008 and 2018, 111 patients received linezolid and 4 of these patients cultured linezolid resistant S. aureus . We sequenced 11 resistant and 21 susceptible isolates from these 4 subjects. Phylogenetic analysis indicated that linezolid resistance developed in ST5 or ST105 backgrounds. Three individuals had linezolid resistant S. aureus with a G2576T mutation in 23S rRNA. One of these subjects additionally had a mutS - mutL - hypermutating S. aureus that produced 5 resistant isolates with multiple ribosomal subunit mutations. In one subject, the genetic basis for linezolid resistance was unclear. Conclusions: Linezolid resistance evolved in 4 of 111 patients in this study. Linezolid resistance occurred by multiple genetic mechanisms. All resistant strains developed in ST5 or ST105 MRSA backgrounds. Key Point: Linezolid resistance arises through multiple genetic mechanisms and could be facilitated by mutator phenotypes. Linezolid resistance was transient, possibly due to growth disadvantage.

Effectiveness and acceptability of intranasal povidone-iodine decolonization among fracture fixation surgery patients to reduce Staphylococcus aureus nasal colonization.

We evaluated povidone-iodine (PVI) decolonization among 51 fracture-fixation surgery patients. PVI was applied twice on the day of surgery. Patients were tested for S. aureus nasal colonization and surveyed. Mean S. aureus concentrations decreased from 3.13 to 1.15 CFU/mL (P = .03). Also, 86% of patients stated that they felt neutral or positive about their PVI experience.

Using nasal povidone-iodine to prevent bloodstream infections and transmission of Staphylococcus aureus among haemodialysis patients: a stepped-wedge cluster randomised control trial protocol.

INTRODUCTION: Approximately 38% of haemodialysis patients carry Staphylococcus aureus in their noses, and carriers have a nearly four-fold increased risk of S. aureus access-related bloodstream infections (BSIs) compared with non-carriers. Our objective is to determine the clinical efficacy and effectiveness of a novel intervention using nasal povidone-iodine (PVI) to prevent BSIs among patients in haemodialysis units. We will survey patients and conduct qualitative interviews with healthcare workers to identify barriers and facilitators to implementing the intervention. METHODS AND ANALYSIS: We will perform an open-label, stepped-wedge cluster randomised trial to assess the effectiveness of nasal PVI compared with standard care. Sixteen outpatient haemodialysis units will participate in the study. The 3-year trial period will be divided into a 4-month baseline period and eight additional 4-month time blocks. The primary outcome of the study will be S. aureus BSI, defined as a S. aureus positive blood culture collected in the outpatient setting or within one calendar day after a hospital admission. The study team will evaluate characteristics of individual patients and the clusters by exposure status (control or intervention) to assess the balance between groups, and calculate descriptive statistics such as average responses separately for control and intervention survey questions. ETHICS AND DISSEMINATION: This study has received IRB approval from all study sites. A Data Safety and Monitoring Board will monitor this multicentre clinical trial. We will present our results at international meetings. The study team will publish findings in peer-reviewed journals and make each accepted peer-reviewed manuscript publicly available. TRIAL REGISTRATION NUMBER: NCT04210505.

MRSA strains with distinct accessory genes predominate at different ages in cystic fibrosis.

RATIONALE: Methicillin resistant Staphylococcus aureus (MRSA) is prevalent and consequential in cystic fibrosis (CF). Whole genome sequencing (WGS) could reveal genomic differences in MRSA associated with poorer outcomes or detect MRSA transmission. OBJECTIVES: To identify MRSA genes associated with low lung function and potential MRSA transmission in CF. METHODS: We collected 97 MRSA isolates from 74 individuals with CF from 2017 and performed short-read WGS. We determined sequence type (ST) and the phylogenetic relationship between isolates. We aligned accessory genes from 25 reference genomes to genome assemblies, classified isolates by accessory gene content, and correlated the accessory genome to clinical outcomes. RESULTS: The most prevalent ST were ST5 (N = 55), ST8 (N = 15), and ST105 (N = 14). Closely related MRSA strains were shared by family members with CF, but rarely between unrelated individuals. Three clusters of MRSA were identified by accessory genome content. Cluster A, including ST5 and ST105, was highly prevalent at all ages. Cluster B, including ST8, was more limited to younger patients. Cluster C included 6 distantly related strains. Patients 20 years old and younger infected with Cluster A had lower forced expiratory volume in the first second (FEV1 ) and higher sputum biomass compared to similar-aged patients with Cluster B. CONCLUSIONS: In this CF cohort, we identified MRSA subtypes that predominate at different ages and differ by accessory gene content. The most prevalent cluster of MRSA, including ST5 and ST105, was associated with lower FEV1 . ST8 MRSA was more common in younger patients and thus has the potential to rise in prevalence as these patients age.

A randomized control trial evaluating efficacy of antimicrobial impregnated hospital privacy curtains in an intensive care setting.

BACKGROUND: Acquisition of pathogens into health care settings from prior room occupants has been documented. Hospital room privacy curtains are at high risk for pathogenic bacterial contamination. Antimicrobial impregnated curtains could be effective in reducing contamination. METHODS: Rooms within an intensive care unit at The University of Iowa Hospitals and Clinics were randomized to 3 arms. The 2 intervention arms: (1) halamine antimicrobial curtains (BioSmart curtain [BSC]) and (2) halamine antimicrobial curtains sprayed twice weekly with a sodium hypochlorite-based disinfecting spray (BSC-pre and BSC-post) and a third control arm (standard curtain [SC]). Samples were collected twice weekly for 3 weeks to assess pathogenic bacterial contamination. RESULTS: The likelihood of remaining uncontaminated was 38% for SC, 37% for BSC, and 60% for the BSC-pre group. Time to event (contamination) analysis found no statistically significant difference between pathogenic contamination between the SC, BSC, and BSC-pre groups (P value = .1921). There was a decrease in average colony count for BSC curtains compared with control, however, this difference was not statistically significant. Hypochlorite spray was found to transiently decontaminate curtains, but effects dissipated after 72 hours. CONCLUSIONS: BSC did not show a significant reduction in pathogenic contamination compared with control. Antimicrobial curtains could have a role in reducing environmental contamination in the health care setting. Future studies should be done to determine the long-term effects of using antimicrobial curtains in health care.

Bioaerosols generated from toilet flushing in rooms of patients with Clostridioides difficile infection.

BACKGROUND: Clostridioides difficile infection (CDI) is the most frequently reported hospital-acquired infection in the United States. Bioaerosols generated during toilet flushing are a possible mechanism for the spread of this pathogen in clinical settings. OBJECTIVE: To measure the bioaerosol concentration from toilets of patients with CDI before and after flushing. DESIGN: In this pilot study, bioaerosols were collected 0.15 m, 0.5 m, and 1.0 m from the rims of the toilets in the bathrooms of hospitalized patients with CDI. Inhibitory, selective media were used to detect C. difficile and other facultative anaerobes. Room air was collected continuously for 20 minutes with a bioaerosol sampler before and after toilet flushing. Wilcoxon rank-sum tests were used to assess the difference in bioaerosol production before and after flushing. SETTING: Rooms of patients with CDI at University of Iowa Hospitals and Clinics. RESULTS: Bacteria were positively cultured from 8 of 24 rooms (33%). In total, 72 preflush and 72 postflush samples were collected; 9 of the preflush samples (13%) and 19 of the postflush samples (26%) were culture positive for healthcare-associated bacteria. The predominant species cultured were Enterococcus faecalis, E. faecium, and C. difficile. Compared to the preflush samples, the postflush samples showed significant increases in the concentrations of the 2 large particle-size categories: 5.0 µm (P = .0095) and 10.0 µm (P = .0082). CONCLUSIONS: Bioaerosols produced by toilet flushing potentially contribute to hospital environmental contamination. Prevention measures (eg, toilet lids) should be evaluated as interventions to prevent toilet-associated environmental contamination in clinical settings.

Correlation between Broth Microdilution and Disk Diffusion Results when Testing Ceftazidime-Avibactam against a Challenge Collection of Enterobacterales Isolates: Results from a Multilaboratory Study.

We assessed the ceftazidime-avibactam disk diffusion breakpoints that provide the lowest discrepancy error rates by testing an Enterobacterales isolate collection with ceftazidime-avibactam MIC values near the breakpoints. Isolates (n = 112) were susceptibility tested by broth microdilution and disk diffusion methods in 3 laboratories. Current disk diffusion breakpoints (≥21/≤20 mm for susceptible/resistant) provided the lowest error rates, but confirmatory MIC testing is indicated for isolates with inhibition zones of 20 to 22 mm.

Candidemia surveillance in Iowa: emergence of echinocandin resistance.

We performed prospective surveillance for candidemia at 14 Iowa hospitals in 2011-2012. A total of 163 episodes were analyzed. Candida albicans (n = 69 [42%]) and Candida glabrata (n = 58 [36%]) were the most common species. Antifungal resistance was uncommon; 9% of C. glabrata were fluconazole resistant, and 5% (3 isolates) were intermediate or resistant to 1 or more of the echinocandins. Molecular analyses of the fks1 and fks2 hotspots of the C. glabrata revealed no mutations except in 2 of these 3 isolates (L628R and S629P in fks1). Compared with previous surveillance performed in 1998-2001, there was a decrease in proportion of candidemia due to C. albicans (58 to 42%) and an increased proportion due to C. glabrata (20 to 36%). Further emergence of echinocandin resistance among the increasingly common species C. glabrata would complicate the management of this life-threatening infection.

Novel hospital curtains with antimicrobial properties: a randomized, controlled trial.

DESIGN: Privacy curtains that separate patient care areas in hospitals may play an important role in the transmission of healthcare-associated pathogens. The aim of this randomized, controlled trial was to assess the effectiveness in a clinical setting of curtains incorporating a complex element compound (CEC) with antimicrobial properties. SETTING: Twenty-one rooms in a surgical intensive care unit (ICU) and 9 rooms in a medical ICU were randomly selected to receive either a new standard curtain or a new identical-looking CEC curtain. Fifteen rooms received CEC curtains and 15 received standard curtains. METHODS: Cultures were performed of samples that were collected from curtains twice a week for 4 weeks (23 days). Contamination was determined according to standard microbiologic methods. Time to contamination was assessed with the Wilcoxon rank-sum test and survival analysis. Incidence rates of contamination were compared using Poisson regression. RESULTS: The median time to first contamination was 7 times longer for CEC curtains than for standard curtains (14 vs 2 days; [Formula: see text]). CEC curtains were significantly less contaminated than standard curtains according to earlier culture results but not significantly different for later culture results. Fourteen CEC curtains and 13 standard curtains were contaminated at least once ([Formula: see text]). The adjusted rate of contamination was 29% lower among CEC versus standard curtains, but this was not statistically significant (rate ratio, 0.71; 95% CI, 0.48-1.07). CONCLUSIONS: CEC privacy curtains increase the time to first contamination as compared with standard curtains. Use of privacy curtains with antimicrobial properties could increase the time between washings and may potentially play a role in decreasing pathogen transmission.

The changing epidemiology of healthcare-associated candidemia over three decades.

We describe the epidemiology of healthcare-associated candidemia (HAC) in our tertiary care hospital, in comparison with both the pre-fluconazole (pre-FLU) and pre-echinocandin (pre-EC) eras. We identified all patients with HAC using microbiology records from 1/2004 to 12/2007, reviewed medical records, and pulled isolates for testing. We compared mortality, underlying illness, Candida species distribution, and antifungal susceptibility with 2 prior University of Iowa cohorts (88 patients from 1983 to 1986 [pre-FLU], and 108 from 1997 to 2001 [pre-EC]). Of 108 patients with HAC from 2004 to 2007, species distribution was 47% C. albicans, 29% C. glabrata, 12% C. parapsilosis, 6% C. tropicalis, and no C. krusei. Compared with pre-FLU and pre-EC eras, there was a reduction in % C. albicans (from 61% and 60%, respectively), an increase in % C. glabrata (from 0% and 16%), and no change in % C. parapsilosis over time (12% and 12%). In-hospital mortality was lower in 2004-2007 than both pre-FLU and pre-EC (31% versus 57-61%), and 30-day mortality was also lower (33% versus 48% in pre-EC). Mean Charlson index was lower for the 2004-2007 cohort than pre-EC (3.0 versus 3.4)-fewer patients had leukemia or lymphoma (8% versus 16%) or other malignancies (18% versus 24%), while more were surgical patients (58% versus 48%). Using the new Clinical and Laboratory Standards Institute breakpoints for FLU and caspofungin, we found no caspofungin resistance, and FLU resistance only among C. glabrata (15% had FLU MICs >32 µg/mL). The epidemiology of HAC is changing at our hospital, with continued emergence of C. glabrata, fewer cases among oncology patients, and lower in-hospital and 30-day mortality.

Hospital privacy curtains are frequently and rapidly contaminated with potentially pathogenic bacteria.

BACKGROUND: Privacy curtains are a potentially important site of bacterial contamination in hospitals. We performed a longitudinal study to determine the prevalence and time course of bacterial contamination on privacy curtains. METHODS: Over a 3-week period, swab cultures (n = 180) were obtained twice weekly from the leading edge of 43 curtains in 30 rooms in 2 intensive care units and a medical ward. Curtains were marked to determine when they were changed. Contamination with Staphylococcus aureus, methicillin-resistant S aureus (MRSA), Enterococcus spp, vancomycin-resistant enterococcus (VRE), or aerobic gram-negative rods was determined by standard microbiologic methods. To distinguish persistence of pathogens on curtains from recontamination, all VRE and MRSA were typed using pulsed-field gel electrophoresis. RESULTS: Twelve of 13 curtains (92%) placed during the study showed contamination within 1 week. Forty-one of 43 curtains (95%) demonstrated contamination on at least 1 occasion, including 21% with MRSA and 42% with VRE. Eight curtains yielded VRE at multiple time points: 3 with persistence of a single isolate type and 5 with different types, suggesting frequent recontamination. CONCLUSION: Privacy curtains are rapidly contaminated with potentially pathogenic bacteria. Further studies should investigate the role of privacy curtains in pathogen transmission and provide interventions to reduce curtain contamination.

Wild-type minimum effective concentration distributions and epidemiologic cutoff values for caspofungin and Aspergillus spp. as determined by Clinical and Laboratory Standards Institute broth microdilution methods.

Antifungal susceptibility testing of Aspergillus spp. against caspofungin has been standardized by the Clinical and Laboratory Standards Institute (CLSI). Recent studies have documented breakthrough infections with Aspergillus spp. for which the minimum effective concentration (MEC) for caspofungin ranged from 0.25 to 8 microg/mL. We tested a collection of 1590 clinical isolates of Aspergillus spp. (188 Aspergillus flavus, 1187 Aspergillus fumigatus, 114 Aspergillus niger, 71 Aspergillus terreus, and 30 Aspergillus versicolor) against caspofungin using the CLSI broth microdilution method. An epidemiologic cutoff value (ECV) of 8 microg/mL [1]), and 1 A. versicolor (4 microg/mL). The establishment of the WT MEC distributions and ECVs for caspofungin and the major species of Aspergillus will be useful in resistance surveillance and is an important step toward the development of clinical breakpoints.

Comparison of results of fluconazole and voriconazole disk diffusion testing for Candida spp. with results from a central reference laboratory in the ARTEMIS DISK Global Antifungal Surveillance Program.

The accuracy of antifungal susceptibility testing is important for reliable resistance surveillance and for the clinical management of patients with serious infections due to Candida spp. Our primary objective was to compare the results of fluconazole and voriconazole disk diffusion testing of 3227 Candida spp. performed by 47 centers participating in the ARTEMIS program with disk diffusion and MIC results obtained by the central reference laboratory. The overall categoric agreement between participant disk diffusion test results and reference MIC results was 87% for fluconazole and 95.2% for voriconazole. Likewise good agreement was observed between participant disk diffusion test results and reference laboratory disk diffusion test results, with an agreement of 90.5%, 1% very major error (VME), and 3.4% major error (ME) for fluconazole and 94.2%, 1.1% VME, and 2.5% ME for voriconazole. The disk diffusion test was reliable for detecting those isolates of Candida spp. that were characterized as resistant to fluconazole and voriconazole by MIC testing. External quality assurance data obtained by surveillance programs such as the ARTEMIS Global Antifungal Surveillance Program ensure the generation of useful surveillance data and result in the continued improvement of antifungal susceptibility testing protocols.

A global evaluation of voriconazole activity tested against recent clinical isolates of Candida spp.

Voriconazole susceptibility testing was performed on 7191 Candida spp. from 78 centers worldwide between 2004 and 2007. Voriconazole was very active in vitro (MIC(50)/MIC(90), 0.008/0.25 microg/mL; 98% susceptible). In comparison to 5866 Candida spp. isolates collected during global surveillance from 1997 to 2001, there were no changes in voriconazole mean MIC or MIC distribution.

Strain-relatedness of methicillin-resistant Staphylococcus aureus isolates recovered from patients with repeated infection.

Invasive disease following methicillin-resistant Staphylococcus aureus (MRSA) detection is common, regardless of whether initial detection involves colonization or infection. We assessed the genetic relatedness of isolates obtained > or =2 weeks apart representing either repeated infections or colonization-infection sets to determine if infections are likely to be caused by previously harbored strains. We found that MRSA infection following initial colonization or infection is caused by the same strain in most cases, suggesting that a single successful attempt at decolonization may prevent the majority of later infection.

Evaluation of disk diffusion and Etest compared to broth microdilution for antifungal susceptibility testing of posaconazole against clinical isolates of filamentous fungi.

We performed Etest, disk diffusion, and broth microdilution susceptibility testing of posaconazole against 146 clinical isolates of filamentous fungi. By using provisional breakpoints for comparison purposes only, categorical agreement between the results of the agar-based methods and those of broth microdilution were 96 to 98%, with no very major errors. These agar-based methods hold promise as simple and reliable methods for determining the posaconazole susceptibilities of filamentous fungi.

Evaluation of Etest and disk diffusion methods compared with broth microdilution antifungal susceptibility testing of clinical isolates of Candida spp. against posaconazole.

We performed Etest, disk diffusion, and broth microdilution susceptibility testing of 2,171 clinical isolates of Candida spp. against posaconazole. By using provisional breakpoints for comparison purposes only, the categorical agreement between the agar-based methods and broth microdilution results ranged from 93 to 98%, with <1% very major errors. The essential agreement (within 2 well dilutions) between the Etest and broth microdilution methods was 94%. These agar-based methods hold promise as simple and reliable methods for determination of the posaconzole susceptibilities of Candida spp.

Caspofungin activity against clinical isolates of fluconazole-resistant Candida.

A total of 7,837 clinical isolates of Candida were tested against fluconazole, and 351 resistant (fluconazole MIC >/=64 micro g/ml) isolates were identified (4% of the total tested). All fluconazole-resistant isolates were inhibited by caspofungin at concentrations that can be exceeded by standard doses (MIC at which 90% of the isolates were inhibited, 1 micro g/ml; 99% of the MICs were