Early introductions and transmission of SARS-CoV-2 variant B.1.1.7 in the United States.

The emergence and spread of SARS-CoV-2 lineage B.1.1.7, first detected in the United Kingdom, has become a global public health concern because of its increased transmissibility. Over 2,500 COVID-19 cases associated with this variant have been detected in the United States (US) since December 2020, but the extent of establishment is relatively unknown. Using travel, genomic, and diagnostic data, we highlight that the primary ports of entry for B.1.1.7 in the US were in New York, California, and Florida. Furthermore, we found evidence for many independent B.1.1.7 establishments starting in early December 2020, followed by interstate spread by the end of the month. Finally, we project that B.1.1.7 will be the dominant lineage in many states by mid- to late March. Thus, genomic surveillance for B.1.1.7 and other variants urgently needs to be enhanced to better inform the public health response.

Fungal microbiota sustains lasting immune activation of neutrophils and their progenitors in severe COVID-19.

Gastrointestinal fungal dysbiosis is a hallmark of several diseases marked by systemic immune activation. Whether persistent pathobiont colonization during immune alterations and impaired gut barrier function has a durable impact on host immunity is unknown. We found that elevated levels of Candida albicans immunoglobulin G (IgG) antibodies marked patients with severe COVID-19 (sCOVID-19) who had intestinal Candida overgrowth, mycobiota dysbiosis and systemic neutrophilia. Analysis of hematopoietic stem cell progenitors in sCOVID-19 revealed transcriptional changes in antifungal immunity pathways and reprogramming of granulocyte myeloid progenitors (GMPs) for up to a year. Mice colonized with C. albicans patient isolates experienced increased lung neutrophilia and pulmonary NETosis during severe acute respiratory syndrome coronavirus-2 infection, which were partially resolved with antifungal treatment or by interleukin-6 receptor blockade. sCOVID-19 patients treated with tocilizumab experienced sustained reductions in C. albicans IgG antibodies titers and GMP transcriptional changes. These findings suggest that gut fungal pathobionts may contribute to immune activation during inflammatory diseases, offering potential mycobiota-immune therapeutic strategies for sCOVID-19 with prolonged symptoms.

Promoter-specific transcription inhibition in Staphylococcus aureus by a phage protein.

Phage G1 gp67 is a 23 kDa protein that binds to the Staphylococcus aureus (Sau) RNA polymerase (RNAP) σ(A) subunit and blocks cell growth by inhibiting transcription. We show that gp67 has little to no effect on transcription from most promoters but is a potent inhibitor of ribosomal RNA transcription. A 2.0-Å-resolution crystal structure of the complex between gp67 and Sau σ(A) domain 4 (σ(A)(4)) explains how gp67 joins the RNAP promoter complex through σ(A)(4) without significantly affecting σ(A)(4) function. Our results indicate that gp67 forms a complex with RNAP at most, if not all, σ(A)-dependent promoters, but selectively inhibits promoters that depend on an interaction between upstream DNA and the RNAP α-subunit C-terminal domain (αCTD). Thus, we reveal a promoter-specific transcription inhibition mechanism by which gp67 interacts with the RNAP promoter complex through one subunit (σ(A)), and selectively affects the function of another subunit (αCTD) depending on promoter usage.

Reporting of Antimicrobial Resistance from Blood Cultures, an Antibacterial Resistance Leadership Group Survey Summary: Resistance Marker Reporting Practices from Positive Blood Cultures.

BACKGROUND: We assessed how laboratories use and handle reporting of results of rapid diagnostics performed on positive blood culture broths, with a focus on antimicrobial resistance (AMR) markers. METHODS: A survey assembled by the Antibacterial Resistance Leadership Group Diagnostics Committee was circulated from December 2020 to May 2021. The survey was sent to local hospitals, shared on the ClinMicroNet and Division C listservs, and included in a College of American Pathologists proficiency testing survey. RESULTS: Ninety-six laboratories of various sizes across the United States (95%) and outside of the United States (5%) participated. Of the laboratories that had at least 1 rapid diagnostic in place (94%), significant heterogeneity in methods used and reporting practices was found across community (52%) and academic (40%) laboratories serving hospitals of various sizes. Respondents had implemented 1 to 6 different panels/platforms for a total of 31 permutations. Methods of reporting rapid organism identification and AMR results varied from listing all targets as "detected"/"not detected" (16-22%) without interpretive guidance, to interpreting results (23-42%), or providing therapeutic guidance comments to patient-facing healthcare teams (3-17%). CONCLUSIONS: Current approaches to reporting molecular AMR test results from positive blood culture vary significantly across clinical laboratories. Providing interpretative comments with therapeutic guidance alongside results reported may assist clinicians who are not well-versed in genetic mechanisms of AMR. However, this is currently not being done in all clinical laboratories. Standardized strategies for AMR gene result reporting are needed.

A multifaceted cellular damage repair and prevention pathway promotes high-level tolerance to ß-lactam antibiotics.

Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two-component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high-level ß-lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that ß-lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the ∆vxrAB mutant. Mutating major iron uptake systems dramatically increases ∆vxrAB tolerance to ß-lactams. We propose that VxrAB reduces antibiotic-induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against ß-lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganism's ability to counteract diverse antibiotic-induced stresses promotes high-level antibiotic tolerance and highlights the complex secondary responses elicited by antibiotics.

Impact of a Rapid Molecular Test for Klebsiella pneumoniae Carbapenemase and Ceftazidime-Avibactam Use on Outcomes After Bacteremia Caused by Carbapenem-Resistant Enterobacterales.

BACKGROUND: Patients with bacteremia due to carbapenem-resistant Enterobacterales (CRE) experience delays until appropriate therapy and high mortality rates. Rapid molecular diagnostics for carbapenemases and new ß-lactam/ß-lactamase inhibitors may improve outcomes. METHODS: We conducted an observational study of patients with CRE bacteremia from 2016 to 2018 at 8 New York and New Jersey medical centers and assessed center-specific clinical microbiology practices. We compared time to receipt of active antimicrobial therapy and mortality between patients whose positive blood cultures underwent rapid molecular testing for the Klebsiella pneumoniae carbapenemase (KPC) gene (blaKPC) and patients whose cultures did not undergo this test. CRE isolates underwent antimicrobial susceptibility testing by broth microdilution and carbapenemase profiling by whole-genome sequencing. We also assessed outcomes when ceftazidime-avibactam and polymyxins were used as targeted therapies. RESULTS: Of 137 patients with CRE bacteremia, 89 (65%) had a KPC-producing organism. Patients whose blood cultures underwent blaKPC PCR testing (n = 51) had shorter time until receipt of active therapy (median: 24 vs 50 hours; P = .009) compared with other patients (n = 86) and decreased 14-day (16% vs 37%; P = .007) and 30-day (24% vs 47%; P = .007) mortality. blaKPC PCR testing was associated with decreased 30-day mortality (adjusted odds ratio: .37; 95% CI: .16-.84) in an adjusted model. The 30-day mortality rate was 10% with ceftazidime-avibactam monotherapy and 31% with polymyxin monotherapy (P = .08). CONCLUSIONS: In a KPC-endemic area, blaKPC PCR testing of positive blood cultures was associated with decreased time until appropriate therapy and decreased mortality for CRE bacteremia, and ceftazidime-avibactam is a reasonable first-line therapy for these infections.

Comparison between Perianal Swab and Stool Specimens for Detecting Colonization with Extended-Spectrum Beta-Lactamase-Producing and Fluoroquinolone-Resistant Enterobacterales.

Stool specimens are frequently used to detect gastrointestinal tract colonization with antimicrobial-resistant enteric bacteria, but they cannot be rapidly collected. Perianal swab specimens can be collected more quickly and efficiently, but data evaluating their suitability as a specimen type for this purpose are sparse. We performed selective culture for extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) and fluoroquinolone-resistant Enterobacterales (FQRE) using paired perianal swab and stool specimens that were collected within 1 day of each other from hematopoietic cell transplant recipients and patients with acute leukemia. Nineteen (7.6%) of 251 stool specimens yielded ESBL-E and 64 (26%) of 246 stool specimens yielded FQRE. The positive percent agreement of perianal swab specimens compared to stool specimens was 95% (18/19; 95% confidence interval [CI], 74% to 100%) for detecting ESBL-E and 95% (61/64; 95% CI, 87% to 99%) for detecting FQRE. The concordance between specimen types was 98% (95% CI, 97% to 100%). Perianal swabs are a reliable specimen type for surveillance of the gastrointestinal tract for ESBL-E and FQRE.

Prevalence and Characterization of the Cefazolin Inoculum Effect in North American Methicillin-Susceptible Staphylococcus aureus Isolates.

Antistaphylococcal penicillins and cefazolin remain the primary treatments for infections with methicillin-susceptible Staphylococcus aureus (MSSA). The cefazolin inoculum effect (CzIE) causes the cefazolin MIC to be elevated in proportion to the number of bacteria in the inoculum. The objective of this multicenter study was to evaluate the prevalence of the CzIE in North American MSSA isolates. Clinical MSSA isolates from six microbiology laboratories in the United States and one microbiology laboratory in Canada were screened for the CzIE by broth microdilution at a standard inoculum (~5 × 105 CFU/mL) and a high inoculum (~5 × 107 CFU/mL). Genome sequencing was performed to further characterize the MSSA isolates. The CzIE was present in 57/305 (18.6%) MSSA isolates, ranging from 0% to 27.9% across study sites. More of the CzIE-positive isolates (29.8%) had standard inoculum cefazolin MICs of 1.0 µg/mL than the CzIE-negative isolates did (3.2%) (P < 0.0001). Conversely, more CzIE-negative isolates (39.5%) had standard inoculum MICs of 0.25 µg/mL than the CzIE positive isolates did (5.3%) (P < 0.0001). The most common BlaZ ß-lactamase types found in the CzIE-positive strains were type C (53.7%) and type A (44.4%). ST8 and ST30 were the most common sequence types among CzIE-positive isolates and correlated with BlaZ type C and A, respectively. The CzIE was present in up to a quarter of clinical MSSA isolates from North American clinical laboratories. Further studies to determine the impact of the presence of the CzIE on clinical outcomes are needed.

Partial ORF1ab Gene Target Failure with Omicron BA.2.12.1.

Mutations in the genome of SARS-CoV-2 can affect the performance of molecular diagnostic assays. In some cases, such as S-gene target failure, the impact can serve as a unique indicator of a particular SARS-CoV-2 variant and provide a method for rapid detection. Here, we describe partial ORF1ab gene target failure (pOGTF) on the cobas SARS-CoV-2 assays, defined by a ≥2-thermocycle delay in detection of the ORF1ab gene compared to that of the E-gene. We demonstrate that pOGTF is 98.6% sensitive and 99.9% specific for SARS-CoV-2 lineage BA.2.12.1, an emerging variant in the United States with spike L452Q and S704L mutations that may affect transmission, infectivity, and/or immune evasion. Increasing rates of pOGTF closely mirrored rates of BA.2.12.1 sequences uploaded to public databases, and, importantly, increasing local rates of pOGTF also mirrored increasing overall test positivity. Use of pOGTF as a proxy for BA.2.12.1 provides faster tracking of the variant than whole-genome sequencing and can benefit laboratories without sequencing capabilities.

The Diagnostic Accuracy Of Procalcitonin for Urinary Tract Infection in Hospitalized Older Adults: a Prospective Study.

BACKGROUND: The diagnosis of urinary tract infection (UTI) is challenging among hospitalized older adults, particularly among those with altered mental status. OBJECTIVE: To determine the diagnostic accuracy of procalcitonin (PCT) for UTI in hospitalized older adults. DESIGN: We performed a prospective cohort study of older adults (≥65 years old) admitted to a single hospital with evidence of pyuria on urinalysis. PCT was tested on initial blood samples. The reference standard was a clinical definition that included the presence of a positive urine culture and any symptom or sign of infection referable to the genitourinary tract. We also surveyed the treating physicians for their clinical judgment and performed expert adjudication of cases for the determination of UTI. PARTICIPANTS: Two hundred twenty-nine study participants at a major academic medical center. MAIN MEASURES: We calculated the area under the receiver operating characteristic curve (AUC) of PCT for the diagnosis of UTI. KEY RESULTS: In this study cohort, 61 (27%) participants met clinical criteria for UTI. The median age of the overall cohort was 82.6 (IQR 74.9-89.7) years. The AUC of PCT for the diagnosis of UTI was 0.56 (95% CI, 0.46-0.65). A series of sensitivity analyses on UTI definition, which included using a decreased threshold for bacteriuria, the treating physicians' clinical judgment, and independent infectious disease specialist adjudication, confirmed the negative result. CONCLUSIONS: Our findings demonstrate that PCT has limited value in the diagnosis of UTI among hospitalized older adults. Clinicians should be cautious using PCT for the diagnosis of UTI in hospitalized older adults.

Impact of Severe Acute Respiratory Syndrome Coronavirus 2 Viral Load on Risk of Intubation and Mortality Among Hospitalized Patients With Coronavirus Disease 2019.

BACKGROUND: Patients hospitalized with coronavirus disease 2019 (COVID-19) frequently require mechanical ventilation and have high mortality rates. However, the impact of viral burden on these outcomes is unknown. METHODS: We conducted a retrospective cohort study of patients hospitalized with COVID-19 from 30 March 2020 to 30 April 2020 at 2 hospitals in New York City. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load was assessed using cycle threshold (Ct) values from a reverse transcription-polymerase chain reaction assay applied to nasopharyngeal swab samples. We compared characteristics and outcomes of patients with high, medium, and low admission viral loads and assessed whether viral load was independently associated with intubation and in-hospital mortality. RESULTS: We evaluated 678 patients with COVID-19. Higher viral load was associated with increased age, comorbidities, smoking status, and recent chemotherapy. In-hospital mortality was 35.0% (Ct <25; n = 220), 17.6% (Ct 25-30; n = 216), and 6.2% (Ct >30; n = 242) with high, medium, and low viral loads, respectively (P < .001). The risk of intubation was also higher in patients with a high viral load (29.1%) compared with those with a medium (20.8%) or low viral load (14.9%; P < .001). High viral load was independently associated with mortality (adjusted odds ratio [aOR], 6.05; 95% confidence interval [CI], 2.92-12.52) and intubation (aOR, 2.73; 95% CI, 1.68-4.44). CONCLUSIONS: Admission SARS-CoV-2 viral load among hospitalized patients with COVID-19 independently correlates with the risk of intubation and in-hospital mortality. Providing this information to clinicians could potentially be used to guide patient care.

Colonization With Fluoroquinolone-Resistant Enterobacterales Decreases the Effectiveness of Fluoroquinolone Prophylaxis in Hematopoietic Cell Transplant Recipients.

BACKGROUND: Levofloxacin prophylaxis is recommended to prevent gram-negative bloodstream infections (BSIs) in patients with prolonged chemotherapy-induced neutropenia. However, increasing fluoroquinolone resistance may decrease the effectiveness of this approach. METHODS: We assessed the prevalence of colonization with fluoroquinolone-resistant Enterobacterales (FQRE) among patients admitted for hematopoietic cell transplantation (HCT) from November 2016 to August 2019 and compared the risk of gram-negative BSI between FQRE-colonized and noncolonized patients. All patients received levofloxacin prophylaxis during neutropenia. Stool samples were collected upon admission for HCT and weekly thereafter until recovery from neutropenia, and underwent selective culture for FQRE. All isolates were identified and underwent antimicrobial susceptibility testing by broth microdilution. FQRE isolates also underwent whole-genome sequencing. RESULTS: Fifty-four of 234 (23%) patients were colonized with FQRE prior to HCT, including 30 of 119 (25%) allogeneic and 24 of 115 (21%) autologous HCT recipients. Recent antibacterial use was associated with FQRE colonization (P = .048). Ninety-one percent of colonizing FQRE isolates were Escherichia coli and 29% produced extended-spectrum ß-lactamases. Seventeen (31%) FQRE-colonized patients developed gram-negative BSI despite levofloxacin prophylaxis, compared to only 2 of 180 (1.1%) patients who were not colonized with FQRE on admission (P < .001). Of the 17 gram-negative BSIs in FQRE-colonized patients, 15 (88%) were caused by FQRE isolates that were genetically identical to the colonizing strain. CONCLUSIONS: Nearly one-third of HCT recipients with pretransplant FQRE colonization developed gram-negative BSI while receiving levofloxacin prophylaxis, and infections were typically caused by their colonizing strains. In contrast, levofloxacin prophylaxis was highly effective in patients not initially colonized with FQRE.

Analysis of Multidrug Resistance in Staphylococcus aureus with a Machine Learning-Generated Antibiogram.

Multidrug resistance (MDR) surveillance consists of reporting MDR prevalence and MDR phenotypes. Detailed knowledge of the specific associations underlying MDR patterns can allow antimicrobial stewardship programs to accurately identify clinically relevant resistance patterns. We applied machine learning and graphical networks to quantify and visualize associations between resistance traits in a set of 1,091 Staphylococcus aureus isolates collected from one New York hospital between 2008 and 2018. Antimicrobial susceptibility testing was performed using reference broth microdilution. The isolates were analyzed by year, methicillin susceptibility, and infection site. Association mining was used to identify resistance patterns that consisted of two or more individual antimicrobial resistance (AMR) traits and quantify the association among the individual resistance traits in each pattern. The resistance patterns captured the majority of the most common MDR phenotypes and reflected previously identified pairwise relationships between AMR traits in S. aureus Associations between ß-lactams and other antimicrobial classes (macrolides, lincosamides, and fluoroquinolones) were common, although the strength of the association among these antimicrobial classes varied by infection site and by methicillin susceptibility. Association mining identified associations between clinically important AMR traits, which could be further investigated for evidence of resistance coselection. For example, in skin and skin structure infections, clindamycin and tetracycline resistance occurred together 1.5 times more often than would be expected if they were independent from one another. Association mining efficiently discovered and quantified associations among resistance traits, allowing these associations to be compared between relevant subsets of isolates to identify and track clinically relevant MDR.

Elevated MICs of Susceptible Antipseudomonal Cephalosporins in Non-Carbapenemase-Producing, Carbapenem-Resistant Pseudomonas aeruginosa: Implications for Dose Optimization.

The present study evaluated the in vitro potency of ceftazidime and cefepime among carbapenem-resistant Pseudomonas aeruginosa isolates collected as part of a global surveillance program and assessed the pharmacodynamic implications using previously published population pharmacokinetics. When susceptible, MICs resulted at the high end of distribution for both ceftazidime and cefepime, thus 6 g/day was required to achieve optimal pharmacodynamic profiles. These findings should be considered in the clinic and for the application of CLSI susceptibility breakpoints.

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.

Evaluation of a Combined Multilocus Sequence Typing and Whole-Genome Sequencing Two-Step Algorithm for Routine Typing of Clostridioides difficile.

Multilocus sequence typing (MLST) is a low-resolution but rapid genotyping method for Clostridioides difficile Whole-genome sequencing (WGS) has emerged as the new gold standard for C. difficile typing, but cost and lack of standardization still limit broad utilization. In this study, we evaluated the potential to combine the portability of MLST with the increased resolution of WGS for a cost-saving approach to routine C. difficile typing. C. difficile strains from two New York City hospitals (hospital A and hospital B) were selected. WGS single-nucleotide polymorphism (wgSNP) was performed using established methods. Sequence types (ST) were determined using PubMLST, while wgSNP analysis was performed using the Bionumerics software. An additional analysis of a subset of data (hospital A) was made comparing the Bionumerics software to the CosmosID pipeline. Cost and turnaround time to results were compared for the algorithmic approach of MLST followed by wgSNP versus direct wgSNP. Among the 202 C. difficile isolates typed, 91% (n = 185/203) clustered within the representative ST, showing a high agreement between MLST and wgSNP. While clustering was similar between the Bionumerics and CosmosID pipelines, large differences in the overall number of SNPs were noted. A two-step algorithm for routine typing results in significantly lower cost than routine use of WGS. Our results suggest that using MLST as a first step in routine typing of C. difficile followed by WGS for MLST concordant strains is a less technically demanding, cost-saving approach for performing C. difficile typing than WGS alone without loss of discriminatory power.

Clinical Performance of the Point-of-Care cobas Liat for Detection of SARS-CoV-2 in 20 Minutes: a Multicenter Study.

Highly accurate testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the point of care (POC) is an unmet diagnostic need in emergency care and time-sensitive outpatient care settings. Reverse transcription-PCR (RT-PCR) technology is the gold standard for SARS-CoV-2 diagnostics. We performed a multisite U.S. study comparing the clinical performance of the first U.S. Food and Drug Administration (FDA)-authorized POC RT-PCR for detection of SARS-CoV-2 in 20 min, the cobas Liat SARS-CoV-2 and influenza A/B nucleic acid test, to the most widely used RT-PCR laboratory test, the cobas 68/8800 SARS-CoV-2 test. Clinical nasopharyngeal swab specimens from 444 patients with 357 evaluable specimens at five U.S. clinical laboratories were enrolled from 21 September 2020 to 23 October 2020. The overall agreement between the Liat and 68/8800 systems for SARS-CoV-2 diagnostics was 98.6% (352/357). Using Liat, positive percent agreement for SARS-CoV-2 was 100% (162/162) and the negative percent agreement was 97.4% (190/195). The Liat is an RT-PCR POC test that provides highly accurate SARS-CoV-2 results in 20 min with performance equivalent to that of high-throughput laboratory molecular testing. Rapid RT-PCR testing at the POC can enable more timely infection control and individual care decisions for coronavirus disease 2019.

Design of a PEGylated Antimicrobial Prodrug with Species-Specific Activation.

The rise of multidrug-resistant (MDR) "superbugs" has created an urgent need to develop new classes of antimicrobial agents to target these organisms. Oligothioetheramides (oligoTEAs) are a unique class of antimicrobial peptide (AMP) mimetics with one promising compound, BDT-4G, displaying potent activity against MDR Pseudomonas aeruginosa clinical isolates. Despite widely demonstrated potency, BDT-4G and other AMP mimetics have yet to enjoy broad preclinical success against systemic infections, primarily due to their cytotoxicity. In this work, we explore a prodrug strategy to render BDT-4G inactive until it is exposed to an enzyme secreted by the targeted bacteria. The prodrug consists of polyethylene glycol (PEG) conjugated to BDT-4G by a peptide substrate. PEG serves to inactivate and reduce the toxicity of BDT-4G by masking its cationic charge and antimicrobial activity is recovered following site-specific cleavage of the short peptide linker by LasA, a virulence factor secreted by P. aeruginosa. This approach concurrently reduces cytotoxicity by greater than 1 order of magnitude in vitro and provides species specificity through the identity of the cleavable linker.

Gut microbiota profiles and fecal beta-glucuronidase activity in kidney transplant recipients with and without post-transplant diarrhea.

Post-transplant diarrhea is a common complication after solid organ transplantation and is frequently attributed to the widely prescribed immunosuppressant mycophenolate mofetil (MMF). Given recent work identifying the relationship between MMF toxicity and gut bacterial ß-glucuronidase activity, we evaluated the relationship between gut microbiota composition, fecal ß-glucuronidase activity, and post-transplant diarrhea. We recruited 97 kidney transplant recipients and profiled the gut microbiota in 273 fecal specimens using 16S rRNA gene sequencing. We further characterized fecal ß-glucuronidase activity in a subset of this cohort. Kidney transplant recipients with post-transplant diarrhea had decreased gut microbial diversity and decreased relative gut abundances of 12 genera when compared to those without post-transplant diarrhea (adjusted p value < .15, Wilcoxon rank sum test). Among the kidney transplant recipients with post-transplant diarrhea, those with higher fecal ß-glucuronidase activity had a more prolonged course of diarrhea (≥7 days) compared to patients with lower fecal ß-glucuronidase activity (91% vs 40%, p = .02, Fisher's exact test). Our data reveal post-transplant diarrhea as a complex phenomenon with decreased gut microbial diversity and commensal gut organisms. This study further links commensal bacterial metabolism with an important clinical outcome measure, suggesting fecal ß-glucuronidase activity could be a novel biomarker for gastrointestinal-related MMF toxicity.

Hydrogel delivery of lysostaphin eliminates orthopedic implant infection by Staphylococcus aureus and supports fracture healing.

Orthopedic implant infections are a significant clinical problem, with current therapies limited to surgical debridement and systemic antibiotic regimens. Lysostaphin is a bacteriolytic enzyme with high antistaphylococcal activity. We engineered a lysostaphin-delivering injectable PEG hydrogel to treat Staphylococcus aureus infections in bone fractures. The injectable hydrogel formulation adheres to exposed tissue and fracture surfaces, ensuring efficient, local delivery of lysostaphin. Lysostaphin encapsulation within this synthetic hydrogel maintained enzyme stability and activity. Lysostaphin-delivering hydrogels exhibited enhanced antibiofilm activity compared with soluble lysostaphin. Lysostaphin-delivering hydrogels eradicated S. aureus infection and outperformed prophylactic antibiotic and soluble lysostaphin therapy in a murine model of femur fracture. Analysis of the local inflammatory response to infections treated with lysostaphin-delivering hydrogels revealed indistinguishable differences in cytokine secretion profiles compared with uninfected fractures, demonstrating clearance of bacteria and associated inflammation. Importantly, infected fractures treated with lysostaphin-delivering hydrogels fully healed by 5 wk with bone formation and mechanical properties equivalent to those of uninfected fractures, whereas fractures treated without the hydrogel carrier were equivalent to untreated infections. Finally, lysostaphin-delivering hydrogels eliminate methicillin-resistant S. aureus infections, supporting this therapy as an alternative to antibiotics. These results indicate that lysostaphin-delivering hydrogels effectively eliminate orthopedic S. aureus infections while simultaneously supporting fracture repair.