Evaluating the Rapid Emergence of Daptomycin Resistance in Corynebacterium: a Multicenter Study.

Members of the genus Corynebacterium are increasingly recognized as pathobionts and can be very resistant to antimicrobial agents. Previous studies have demonstrated that Corynebacterium striatum can rapidly develop high-level daptomycin resistance (HLDR) (MIC, ≥256 µg/ml). Here, we conducted a multicenter study to assay for this in vitro phenotype in diverse Corynebacterium species. Corynebacterium clinical isolates (n = 157) from four medical centers were evaluated. MIC values to daptomycin, vancomycin, and telavancin were determined before and after overnight exposure to daptomycin to identify isolates able to rapidly develop daptomycin nonsusceptibility. To investigate assay reproducibility, 18 isolates were evaluated at three study sites. In addition, the stability of daptomycin nonsusceptibility was tested using repeated subculture without selective pressure. The impact of different medium brands was also investigated. Daptomycin nonsusceptibility emerged in 12 of 23 species evaluated in this study (C. afermentans, C. amycolatum, C. aurimucosum, C. bovis, C. jeikeium, C. macginleyi, C. pseudodiphtheriticum, C. resistens, C. simulans, C. striatum, C. tuberculostearicum, and C. ulcerans) and was detected in 50 of 157 (31.8%) isolates tested. All isolates displayed low (susceptible) MIC values to vancomycin and telavancin before and after daptomycin exposure. Repeated subculture demonstrated that 2 of 9 isolates (22.2%) exhibiting HLDR reverted to a susceptible phenotype. Of 30 isolates tested on three medium brands, 13 (43.3%) had differences in daptomycin MIC values between brands. Multiple Corynebacterium species can rapidly develop daptomycin nonsusceptibility, including HLDR, after a short daptomycin exposure period.

Candida albicans biofilm-induced vesicles confer drug resistance through matrix biogenesis.

Cells from all kingdoms of life produce extracellular vesicles (EVs). Their cargo is protected from the environment by the surrounding lipid bilayer. EVs from many organisms have been shown to function in cell-cell communication, relaying signals that impact metazoan development, microbial quorum sensing, and pathogenic host-microbe interactions. Here, we have investigated the production and functional activities of EVs in a surface-associated microbial community or biofilm of the fungal pathogen Candida albicans. Crowded communities like biofilms are a context in which EVs are likely to function. Biofilms are noteworthy because they are encased in an extracellular polymeric matrix and because biofilm cells exhibit extreme tolerance to antimicrobial compounds. We found that biofilm EVs are distinct from those produced by free-living planktonic cells and display strong parallels in composition to biofilm matrix material. The functions of biofilm EVs were delineated with a panel of mutants defective in orthologs of endosomal sorting complexes required for transport (ESCRT) subunits, which are required for normal EV production in diverse eukaryotes. Most ESCRT-defective mutations caused reduced biofilm EV production, reduced matrix polysaccharide levels, and greatly increased sensitivity to the antifungal drug fluconazole. Matrix accumulation and drug hypersensitivity of ESCRT mutants were reversed by addition of wild-type (WT) biofilm EVs. Vesicle complementation showed that biofilm EV function derives from specific cargo proteins. Our studies indicate that C. albicans biofilm EVs have a pivotal role in matrix production and biofilm drug resistance. Biofilm matrix synthesis is a community enterprise; prior studies of mixed cell biofilms have demonstrated extracellular complementation. Therefore, EVs function not only in cell-cell communication but also in the sharing of microbial community resources.

Community participation in biofilm matrix assembly and function.

Biofilms of the fungus Candida albicans produce extracellular matrix that confers such properties as adherence and drug resistance. Our prior studies indicate that the matrix is complex, with major polysaccharide constituents being α-mannan, ß-1,6 glucan, and ß-1,3 glucan. Here we implement genetic, biochemical, and pharmacological approaches to unravel the contributions of these three constituents to matrix structure and function. Interference with synthesis or export of any one polysaccharide constituent altered matrix concentrations of each of the other polysaccharides. Each of these was also required for matrix function, as assessed by assays for sequestration of the antifungal drug fluconazole. These results indicate that matrix biogenesis entails coordinated delivery of the individual matrix polysaccharides. To understand whether coordination occurs at the cellular level or the community level, we asked whether matrix-defective mutant strains could be coaxed to produce functional matrix through biofilm coculture. We observed that mixed biofilms inoculated with mutants containing a disruption in each polysaccharide pathway had restored mature matrix structure, composition, and biofilm drug resistance. Our results argue that functional matrix biogenesis is coordinated extracellularly and thus reflects the cooperative actions of the biofilm community.

The Extracellular Matrix of Fungal Biofilms.

A key feature of biofilms is their production of an extracellular matrix. This material covers the biofilm cells, providing a protective barrier to the surrounding environment. During an infection setting, this can include such offenses as host cells and products of the immune system as well as drugs used for treatment. Studies over the past two decades have revealed the matrix from different biofilm species to be as diverse as the microbes themselves. This chapter will review the composition and roles of matrix from fungal biofilms, with primary focus on Candida species, Saccharomyces cerevisiae, Aspergillus fumigatus, and Cryptococcus neoformans. Additional coverage will be provided on the antifungal resistance proffered by the Candida albicans matrix, which has been studied in the most depth. A brief section on the matrix produced by bacterial biofilms will be provided for comparison. Current tools for studying the matrix will also be discussed, as well as suggestions for areas of future study in this field.

Evaluation of a Multiplex Bead Assay against Single-Target Assays for Detection of IgG Antibodies to SARS-CoV-2.

Serological assays for SARS-CoV-2 antibodies must be validated for performance with a large panel of clinical specimens. Most existing assays utilize a single antigen target and may be subject to reduced diagnostic specificity. This study evaluated a multiplex assay that detects antibodies to three SARS-CoV-2 targets. Human serum specimens (n = 323) with known previous SARS-CoV-2 exposure status were tested on a commercially available multiplex bead assay (MBA) measuring IgG to SARS-CoV-2 spike protein receptor-binding domain (RBD), nucleocapsid protein (NP), and RBD/NP fusion antigens. Assay performance was evaluated against reverse transcriptase PCR (RT-PCR) results and also compared with test results for two single-target commercial assays. The MBA had a diagnostic sensitivity of 89.8% and a specificity of 100%, with serum collection at >28 days following COVID-19 symptom onset showing the highest seropositivity rates (sensitivity: 94.7%). The MBA performed comparably to single-target assays with the ability to detect IgG against specific antigen targets, with 19 (5.9%) discrepant specimens compared to the NP IgG assay and 12 (3.7%) compared to the S1 RBD IgG assay (kappa coefficients 0.92 and 0.88 compared to NP IgG and S1 RBD IgG assays, respectively. These findings highlight inherent advantages of using a SARS-CoV-2 serological test with multiple antigen targets; specifically, the ability to detect IgG against RBD and NP antigens simultaneously. In particular, the 100.0% diagnostic specificity exhibited by the MBA in this study is important for its implementation in populations with low SARS-CoV-2 seroprevalence or where background antibody reactivity to SARS-CoV-2 antigens has been detected. IMPORTANCE Reporting of SARS-CoV-2 infections through nucleic acid or antigen based diagnostic tests severely underestimates the true burden of exposure in a population. Serological data assaying for antibodies against SARS-CoV-2 antigens offers an alternative source of data to estimate population exposure, but most current immunoassays only include a single target for antibody detection. This report outlines a direct comparison of a multiplex bead assay to two other commercial single-target assays in their ability to detect IgG against SARS-CoV-2 antigens. Against a well-defined panel of 323 serum specimens, diagnostic sensitivity and specificity were very high for the multiplex assay, with strong agreement in IgG detection for single targets compared to the single-target assays. Collection of more data for individual- and population-level seroprofiles allows further investigation into more accurate exposure estimates and research into the determinants of infection and convalescent responses.

More than Just Contaminants: Frequency and Characterization of Polymicrobial Blood Cultures from a Central Clinical Microbiology Laboratory Serving a Large Healthcare System.

BACKGROUND: Polymicrobial blood stream infection is often considered uncommon, and corresponding cultures may be assumed to represent contamination. Here we characterized the prevalence and epidemiology of these cultures submitted to a central clinical microbiology laboratory. METHODS: Blood cultures from 2017 to 2018 (n = 104 547) were evaluated. Polymicrobial blood cultures were defined by the presence of more than one organism in a blood culture set (set = one aerobic and one anaerobic bottle). Data were stratified by patient location and characteristics of the microbiota detected. RESULTS: Of all blood culture sets, 14 600 (14.0%) were positive. Among these, 1651 sets (11.3% of positive cultures; 1.6% of total cultures) were polymicrobial. Most cultures (85.2%) grew two microorganisms; the greatest number of microorganisms in a culture was 6. The most common microorganism pairs were (a) two coagulase-negative staphylococci (CoNS), (b) Corynebacterium and CoNS, and (c) S. aureus and CoNS. Microorganisms in polymicrobial cultures represented microbiota from skin (46.1%), the gastrointestinal (GI) tract (33.9%), strict anaerobes (1.4%), and "other" microorganisms (18.6%). Most cultures with GI microbiota originated from an adult academic medical center compared to community or pediatric settings (40.5% of polymicrobial cultures vs 27.2% and 25.8%, P < 0.0001). Within the academic medical center, patients in an intensive care unit (ICU) or who had bone marrow transplants (BMT) had more cultures suggestive of GI microbiota compared to those from the emergency department (ED) (50.8% and 52.8% vs 31.2%, P < 0.0001). CONCLUSIONS: Polymicrobial blood cultures are common in a variety of healthcare settings and the recovered microorganisms can be clinically relevant.

Evolution of the complex transcription network controlling biofilm formation in Candida species.

We examine how a complex transcription network composed of seven 'master' regulators and hundreds of target genes evolved over a span of approximately 70 million years. The network controls biofilm formation in several Candida species, a group of fungi that are present in humans both as constituents of the microbiota and as opportunistic pathogens. Using a variety of approaches, we observed two major types of changes that have occurred in the biofilm network since the four extant species we examined last shared a common ancestor. Master regulator 'substitutions' occurred over relatively long evolutionary times, resulting in different species having overlapping but different sets of master regulators of biofilm formation. Second, massive changes in the connections between the master regulators and their target genes occurred over much shorter timescales. We believe this analysis is the first detailed, empirical description of how a complex transcription network has evolved.

Urinary Tract Infection With Gram-Positive Bacteria Does Not Cause False-Positive Results with the Urine-Based Human Chorionic Gonadotropic Point-of-Care Assay.

BACKGROUND: False-positive results for human chorionic gonadotropic (hCG) on point-of-care (POC) devices can occur for a variety of technical and biological reasons. It has been postulated that urinary tract infection can result in false-positive POC hCG assays, but the cause of this phenomenon remains elusive. Gram-positive bacteria have previously been reported to express an hCG-like molecule. We investigated whether urinary tract infection with Gram-positive bacteria can result in false-positive POC hCG. METHODS: We utilized remnant clinical urine specimens that had been submitted for culture as part of evaluation for urinary tract infection. Urine specimens with >100,000 colony-forming units per milliliter of Gram-positive bacteria (n = 95) were tested on ICON 20 POC hCG tests (Beckman Coulter). Specimens from adult patients that had been collected for clinical testing in the prior 48 hours were included in the study, and only 1 specimen per patient was included. RESULTS: Of 95 patients with Gram-positive urine specimens, 42 (44%) were female, and the median age was 62 years. The most common bacteria identified during clinical urine culture of these patients' specimens were coagulase-negative Staphylococcus species (36/95, 38%), Enterococcus species (34/95, 36%), and Streptococcus agalactiae (9/95, 9%). Five of 95 (5.3%) urine specimens were positive for POC hCG. Chart review revealed that 3 specimens were from pregnant women and 2 were from patients with cancer diagnoses. CONCLUSIONS: Urine specimens from patients suspected to have urinary tract infection with Gram-positive organisms did not cause positive results on POC hCG test devices.

Evaluating carbapenem restriction practices at a private hospital in Manila, Philippines as a strategy for antimicrobial stewardship.

BACKGROUND: Hospital antimicrobial stewardship programs are especially critical in countries such as the Philippines, where antibiotic resistant infections are highly prevalent. At the study institution in Manila, Philippines, a Prior Approval for Restricted Antimicrobials (PARA) is required for non-infectious disease specialists to prescribe certain antimicrobials, including carbapenems. PARA request forms include specification of empiric or definitive therapy based on diagnostic tests. Recommended duration of therapy is typically 3 days for empiric use and 7 days for definitive, with possible extension upon specialist approval. METHODS: The study took place at an 800-bed tertiary hospital. We performed a retrospective review of patient medical records and laboratory reports dating from January 1 to December 31, 2016. Information related to patient demographics, carbapenem prescription, laboratory diagnosis, and therapy were compiled. Carbapenem prescriptions were classified as 'adherent' or 'non-adherent' according to clinical guidelines related to infection diagnosis, treatment duration, and de-escalation. RESULTS: Of the 185 patients on carbapenem therapy, Prescriptions of carbapenems were either definitive (n = 56), empiric (n = 127), or prophylactic (n = 2) as defined by the ordering provider. 69 out of 185 (37%) prescriptions were deemed non-adherent to guidelines, despite receiving approvals for their respective requests. Of these, 72% were non-adherent due to failure to de-escalate the carbapenem and 28% were non-adherent due to an incomplete course of therapy. CONCLUSION: Despite initial PARA approval for carbapenem therapy, 37% of prescriptions were non-guideline-adherent, highlighting the ongoing challenges in implementing this type of stewardship strategy. In order to increase the effectiveness of PARA, additional approaches may be warranted, including the application of strict policies which reinforce follow-up of available culture results, justification of therapy extension, or referral to an infectious disease specialist.

Multicenter Study Demonstrates Standardization Requirements for Mold Identification by MALDI-TOF MS.

OBJECTIVES: Rapid and accurate mold identification is critical for guiding therapy for mold infections. MALDI-TOF MS has been widely adopted for bacterial and yeast identification; however, few clinical laboratories have applied this technology for routine mold identification due to limited database availability and lack of standardized processes. Here, we evaluated the versatility of the NIH Mold Database in a multicenter evaluation. METHODS: The NIH Mold Database was evaluated by eight US academic centers using a solid media extraction method and a challenge set of 80 clinical mold isolates. Multiple instrument parameters important for spectra optimization were evaluated, leading to the development of two specialized acquisition programs (NIH method and the Alternate-B method). RESULTS: A wide range in performance (33-77%) was initially observed across the eight centers when routine spectral acquisition parameters were applied. Use of the NIH or the Alternate-B specialized acquisition programs, which are different than those used routinely for bacterial and yeast spectral acquisition (MBT_AutoX), in combination with optimized instrument maintenance, improved performance, illustrating that acquisition parameters may be one of the key limiting variable in achieving successful performance. CONCLUSION: Successful mold identification using the NIH Database for MALDI-TOF MS on Biotyper systems was demonstrated across multiple institutions for the first time following identification of critical program parameters combined with instrument optimization. This significantly advances our potential to implement MALDI-TOF MS for mold identification across many institutions. Because instrument variability is inevitable, development of an instrument performance standard specific for mold spectral acquisition is suggested to improve reproducibility across instruments.

Infection control at an urban hospital in Manila, Philippines: a systems engineering assessment of barriers and facilitators.

BACKGROUND: Healthcare facilities in low- and middle-income countries, including the Philippines, face substantial challenges in achieving effective infection control. Early stages of interventions should include efforts to understand perceptions held by healthcare workers who participate in infection control programs. METHODS: We performed a qualitative study to examine facilitators and barriers to infection control at an 800-bed, private, tertiary hospital in Manila, Philippines. Semi-structured interviews were conducted with 22 nurses, physicians, and clinical pharmacists using a guide based on the Systems Engineering Initiative for Patient Safety (SEIPS). Major facilitators and barriers to infection control were reported for each SEIPS factor: person, organization, tasks, physical environment, and technology and tools. RESULTS: Primary facilitators included a robust, long-standing infection control committee, a dedicated infection control nursing staff, and innovative electronic hand hygiene surveillance technology. Barriers included suboptimal dissemination of hand hygiene compliance data, high nursing turnover, clinical time constraints, and resource limitations that restricted equipment purchasing. CONCLUSIONS: The identified facilitators and barriers may be used to prioritize possible opportunities for infection control interventions. A systems engineering approach is useful for conducting a comprehensive work system analysis, and maximizing resources to overcome known barriers to infection control in heavily resource-constrained settings.

Transcriptional rewiring over evolutionary timescales changes quantitative and qualitative properties of gene expression.

Evolutionary changes in transcription networks are an important source of diversity across species, yet the quantitative consequences of network evolution have rarely been studied. Here we consider the transcriptional 'rewiring' of the three GAL genes that encode the enzymes needed for cells to convert galactose to glucose. In Saccharomyces cerevisiae, the transcriptional regulator Gal4 binds and activates these genes. In the human pathogen Candida albicans (which last shared a common ancestor with S. cerevisiae some 300 million years ago), we show that different regulators, Rtg1 and Rtg3, activate the three GAL genes. Using single-cell dynamics and RNA-sequencing, we demonstrate that although the overall logic of regulation is the same in both species-the GAL genes are induced by galactose-there are major differences in both the quantitative response of these genes to galactose and in the position of these genes in the overall transcription network structure of the two species.

A histone deacetylase complex mediates biofilm dispersal and drug resistance in Candida albicans.

UNLABELLED: Biofilms are resilient, surface-associated communities of cells with specialized properties (e.g., resistance to drugs and mechanical forces) that are distinct from those of suspension (planktonic) cultures. Biofilm formation by the opportunistic human fungal pathogen Candida albicans is medically relevant because C. albicans infections are highly correlated with implanted medical devices, which provide efficient substrates for biofilm formation; moreover, biofilms are inherently resistant to antifungal drugs. Biofilms are also important for C. albicans to colonize diverse niches of the human host. Here, we describe four core members of a conserved histone deacetylase complex in C. albicans (Set3, Hos2, Snt1, and Sif2) and explore the effects of their mutation on biofilm formation. We find that these histone deacetylase complex members are needed for proper biofilm formation, including dispersal of cells from biofilms and multifactorial drug resistance. Our results underscore the importance of the physical properties of biofilms in contributing to drug resistance and dispersal and lay a foundation for new strategies to target biofilm dispersal as a potential antifungal intervention. IMPORTANCE: Through the formation of biofilms--surface-associated communities of cells--microorganisms can establish infections, become drug resistant, and evade the host immune system. Here we investigate how four core members of a conserved histone deacetylase complex mediate biofilm formation by Candida albicans, the major fungal pathogen of humans. We show that this histone deacetylase complex is required for biofilm dispersal, a process through which cells leave the biofilm to establish new infections. We also show that the deacetylase complex mediates biofilm drug resistance. This work provides new insight into how the physical properties of biofilms affect dispersal and drug resistance and suggests new potential antifungal strategies that could be effective against biofilms.

Mechanisms of Candida biofilm drug resistance.

Candida commonly adheres to implanted medical devices, growing as a resilient biofilm capable of withstanding extraordinarily high antifungal concentrations. As currently available antifungals have minimal activity against biofilms, new drugs to treat these recalcitrant infections are urgently needed. Recent investigations have begun to shed light on the mechanisms behind the profound resistance associated with the biofilm mode of growth. This resistance appears to be multifactorial, involving both mechanisms similar to conventional, planktonic antifungal resistance, such as increased efflux pump activity, as well as mechanisms specific to the biofilm lifestyle. A unique biofilm property is the production of an extracellular matrix. Two components of this material, ß-glucan and extracellular DNA, promote biofilm resistance to multiple antifungals. Biofilm formation also engages several stress response pathways that impair the activity of azole drugs. Resistance within a biofilm is often heterogeneous, with the development of a subpopulation of resistant persister cells. In this article we review the molecular mechanisms underlying Candida biofilm antifungal resistance and their relative contributions during various growth phases.

Regulatory role of glycerol in Candida albicans biofilm formation.

UNLABELLED: Biofilm formation by Candida albicans on medically implanted devices poses a significant clinical challenge. Here, we compared biofilm-associated gene expression in two clinical C. albicans isolates, SC5314 and WO-1, to identify shared gene regulatory responses that may be functionally relevant. Among the 62 genes most highly expressed in biofilms relative to planktonic (suspension-grown) cells, we were able to recover insertion mutations in 25 genes. Twenty mutants had altered biofilm-related properties, including cell substrate adherence, cell-cell signaling, and azole susceptibility. We focused on one of the most highly upregulated genes in our biofilm proles, RHR2, which specifies the glycerol biosynthetic enzyme glycerol-3-phosphatase. Glycerol is 5-fold-more abundant in biofilm cells than in planktonic cells, and an rhr2Δ/Δ strain accumulates 2-fold-less biofilm glycerol than does the wild type. Under in vitro conditions, the rhr2Δ/Δ mutant has reduced biofilm biomass and reduced adherence to silicone. The rhr2Δ/Δ mutant is also severely defective in biofilm formation in vivo in a rat catheter infection model. Expression profiling indicates that the rhr2Δ/Δ mutant has reduced expression of cell surface adhesin genes ALS1, ALS3, and HWP1, as well as many other biofilm-upregulated genes. Reduced adhesin expression may be the cause of the rhr2Δ/Δ mutant biofilm defect, because overexpression of ALS1, ALS3, or HWP1 restores biofilm formation ability to the mutant in vitro and in vivo. Our findings indicate that internal glycerol has a regulatory role in biofilm gene expression and that adhesin genes are among the main functional Rhr2-regulated genes. IMPORTANCE: Candida albicans is a major fungal pathogen, and infection can arise from the therapeutically intractable biofilms that it forms on medically implanted devices. It stands to reason that genes whose expression is induced during biofilm growth will function in the process, and our analysis of 25 such genes confirms that expectation. One gene is involved in synthesis of glycerol, a small metabolite that we find is abundant in biofilm cells. The impact of glycerol on biofilm formation is regulatory, not solely metabolic, because it is required for expression of numerous biofilm-associated genes. Restoration of expression of three of these genes that specify cell surface adhesins enables the glycerol-synthetic mutant to create a biofilm. Our findings emphasize the significance of metabolic pathways as therapeutic targets, because their disruption can have both physiological and regulatory consequences.