Training and Sustaining: Training and Learning Collaborative Outcomes Across a Statewide Network for Early Autism Diagnosis.

OBJECTIVE: The objective of this study was to describe the development of a primary care professional (PCP) autism diagnosis training model and to report on outcomes related to PCP training and sustained engagement in a longitudinal learning collaborative. METHODS: We developed Accelerating the Diagnosis of Autism with Primary care Training (ADAPT), a training program to prepare PCPs to develop independent competency in evaluation of autism in children aged 14 to 48 months. ADAPT includes didactic and case-based modules and practice-based coaching delivered by an expert diagnostic specialist; after training, PCPs participate in a longitudinal learning collaborative. Aligned with competency-based medical education standards, measures of autism evaluation knowledge and diagnostic competency are collected. RESULTS: From 2021 to 2023, 13 PCPs completed ADAPT didactic and practicum training to reach competency in independent autism evaluation. Clinicians demonstrated significant improvement in total autism knowledge after didactic training (p = 0.02). Scoring agreement on an autism observational assessment tool between clinicians and expert diagnosticians improved over case observations and practicum evaluations. Similarly, PCPs demonstrated improved evaluation competence, moving on average from Advanced Beginner to Competent Performer as rated by expert diagnosticians. After training, PCPs attended 57% of monthly learning collaborative sessions. CONCLUSION: Training PCPs to deliver autism evaluations for young children as part of tiered community-based models of care is a promising solution to address access and waitlist challenges. ADAPT is an intensive, standardized PCP training model that results in achievement of independent competency and sustained engagement in autism evaluation. Effectiveness-implementation studies are needed to ensure scalability and sustainability of training models.

Clostridioides difficile MreE (PBP2) variants facilitate clinical disease during cephalosporin exposures.

Cephalosporins are the most common triggers of healthcare-associated Clostridioides difficile infections (CDI). Here, we confirm gene-level drivers of cephalosporin resistance and their roles in promoting disease. Genomic-epidemiologic analyses of 306 C. difficile isolates from a hospital surveillance program monitoring asymptomatic carriers and CDI patients identified prevalent third-generation cephalosporin resistance to ceftriaxone at >256 ug/mL in 26% of isolates. Resistance was associated with patient cephalosporin exposures 8-10 days before C. difficile detection. Genomic analyses identified variants in the mreE penicillin binding protein 2 (PBP2) associated with resistance to multiple beta-lactam classes. Transfer of variants into susceptible strain CD630 elevated resistance to first and third-generation cephalosporins. Transfer into the mouse-infective strain ATCC 43255 enabled disease when mice were exposed to 500ug/mL cefoperazone, a dose that inhibited the isogenic susceptible strain. Our findings establish roles of cephalosporins and mreE-cephalosporin-resistant variants in CDI and provide testable genetic loci for detecting resistance in patient strains.

Impact of microbiota and host immunologic response on the efficacy of anticholinergic treatment for urgency urinary incontinence.

INTRODUCTION AND HYPOTHESIS: Studies within the past decade have suggested associations among composition of the urinary microbiota, local immune responses, and urinary incontinence symptoms. To investigate these relationships, we evaluated the structure of the urinary microbiome, local inflammatory markers, and patient responses prior to and at 6-weeks after treatment with anticholinergic medication for urgency urinary incontinence (UUI). METHODS: Using a prospective pilot study, we enrolled women who presented with UUI symptoms and were prescribed treatment with anticholinergics. Catheterized urine samples were collected from participants at their baseline and 6-week follow-up visits for microbiological (standard and 16S rRNA gene phylotyping analyses) and cytokine analysis along with the UDI-6 questionnaire and 2-day bladder diary. RESULTS: Patients were Caucasian, post- menopausal, with a median age of 64 and median BMI of 30.1 kg/m2. Among the patients, 75% had UUI symptoms for less than 2 years, but with a frequency of at least a few times a week or every day. Most women were prescribed 10 mg oxybutynin ER daily at enrollment. Patients had varied urinary microbiota by culture and 16S phylotyping, with species of Lactobacillus being the most common, in six samples, in addition to taxa associated with Enterococcus, Staphylococcus, and mixed flora. Cytokine levels showed no differences before and after treatment with anticholinergics, nor correlation with urinary bacteria or microbiome composition. CONCLUSIONS: Our pilot study suggests factors in addition to the urinary microbiome and local immune responses may be involved in patients' response to anticholinergics for UUI.

Gut metabolites predict Clostridioides difficile recurrence.

BACKGROUND: Clostridioides difficile infection (CDI) is the most common hospital acquired infection in the USA, with recurrence rates > 15%. Although primary CDI has been extensively linked to gut microbial dysbiosis, less is known about the factors that promote or mitigate recurrence. Moreover, previous studies have not shown that microbial abundances in the gut measured by 16S rRNA amplicon sequencing alone can accurately predict CDI recurrence. RESULTS: We conducted a prospective, longitudinal study of 53 non-immunocompromised participants with primary CDI. Stool sample collection began pre-CDI antibiotic treatment at the time of diagnosis, and continued up to 8 weeks post-antibiotic treatment, with weekly or twice weekly collections. Samples were analyzed using (1) 16S rRNA amplicon sequencing, (2) liquid chromatography/mass-spectrometry metabolomics measuring 1387 annotated metabolites, and (3) short-chain fatty acid profiling. The amplicon sequencing data showed significantly delayed recovery of microbial diversity in recurrent participants, and depletion of key anaerobic taxa at multiple time-points, including Clostridium cluster XIVa and IV taxa. The metabolomic data also showed delayed recovery in recurrent participants, and moreover mapped to pathways suggesting distinct functional abnormalities in the microbiome or host, such as decreased microbial deconjugation activity, lowered levels of endocannabinoids, and elevated markers of host cell damage. Further, using predictive statistical/machine learning models, we demonstrated that the metabolomic data, but not the other data sources, can accurately predict future recurrence at 1 week (AUC 0.77 [0.71, 0.86; 95% interval]) and 2 weeks (AUC 0.77 [0.69, 0.85; 95% interval]) post-treatment for primary CDI. CONCLUSIONS: The prospective, longitudinal, and multi-omic nature of our CDI recurrence study allowed us to uncover previously unrecognized dynamics in the microbiome and host presaging recurrence, and, in particular, to elucidate changes in the understudied gut metabolome. Moreover, we demonstrated that a small set of metabolites can accurately predict future recurrence. Our findings have implications for development of diagnostic tests and treatments that could ultimately short-circuit the cycle of CDI recurrence, by providing candidate metabolic biomarkers for diagnostics development, as well as offering insights into the complex microbial and metabolic alterations that are protective or permissive for recurrence. Video Abstract.

Inter-species geographic signatures for tracing horizontal gene transfer and long-term persistence of carbapenem resistance.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole-genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms. METHODS: To overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem-susceptible Enterobacterales, isolated from patients from four US hospitals over nearly 5 years. RESULTS: Our CRE collection comprised a diverse range of species, lineages, and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment. CONCLUSIONS: Collectively, the framework we developed provides a clearer, high-resolution picture of the epidemiology of antibiotic resistance importation, spread, and persistence in patients and healthcare networks.

Protocol for using negative pressure isolator systems to study BSL-2 organisms in gnotobiotic murine models.

Here, we present a protocol for the use of negative pressure isolator systems to maintain defined association and contain BSL-2 pathogens in germ-free and gnotobiotic mouse studies. We describe setup and operation of negative pressure isolators with integrated microbiologic procedures, using the BSL-2 pathogen Clostridioides difficile as a working example. This approach supports experimental systems with defined-association mice and enables high-resolution mechanistic studies of pathogen-commensal interactions and their impacts on host phenotypes. For complete details on the use and execution of this protocol, please refer to Girinathan et al. (2021).

In vivo commensal control of Clostridioides difficile virulence.

Leveraging systems biology approaches, we illustrate how metabolically distinct species of Clostridia protect against or worsen Clostridioides difficile infection in mice by modulating the pathogen's colonization, growth, and virulence to impact host survival. Gnotobiotic mice colonized with the amino acid fermenter Paraclostridium bifermentans survive infection with reduced disease severity, while mice colonized with the butyrate-producer, Clostridium sardiniense, succumb more rapidly. Systematic in vivo analyses revealed how each commensal alters the gut-nutrient environment to modulate the pathogen's metabolism, gene regulatory networks, and toxin production. Oral administration of P. bifermentans rescues conventional, clindamycin-treated mice from lethal C. difficile infection in a manner similar to that of monocolonized animals, thereby supporting the therapeutic potential of this commensal species. Our findings lay the foundation for mechanistically informed therapies to counter C. difficile disease using systems biology approaches to define host-commensal-pathogen interactions in vivo.

Conjunctival Commensal Isolation and Identification in Mice.

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an "in-lab" made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Genomic Determination of Relative Risks for Clostridioides difficile Infection From Asymptomatic Carriage in Intensive Care Unit Patients.

BACKGROUND: Clostridioides difficile infections (CDIs) are among the most prevalent hospital-associated infections (HAIs), particularly for intensive care unit (ICU) patients. The risks for developing active CDI from asymptomatic carriage of C. difficile are not well understood. METHODS: We identified asymptomatic C. difficile carriage among 1897 ICU patients using rectal swabs from an existing ICU vancomycin-resistant enterococci (VRE) surveillance program. C. difficile isolates from VRE swabs, and from C. difficile-positive stool samples, were genome sequenced. Spatial-temporal data from hospital records assessed genomically identified clusters for potential transmission events. RESULTS: Genomic analyses identified a diverse set of strains in infected patients and asymptomatic carriers. A total of 7.4% of ICU patients asymptomatically carried C. difficile; 69% of isolates carried an intact toxin locus. In contrast, 96% of C. difficile stool isolates were toxin encoding. CDI rates in asymptomatic carriers of toxin-encoding strains were 5.3% versus 0.57% in noncarriers. The relative risk for CDI with asymptomatic carriage of a toxin-encoding strain was 9.32 (95% confidence interval, 3.25-26.7). Genomic identification of clonal clusters supported analyses for asymptomatic transmission events, with spatial-temporal overlaps identified in 13 of 28 cases. CONCLUSIONS: Our studies provide the first genomically confirmed assessments of CDI relative risk from asymptomatic carriage of toxin-encoding strains and highlight the complex dynamics of asymptomatic transmission in ICUs. Asymptomatic carriers are an active reservoir of C. difficile in the nosocomial environment. C. difficile screening can be implemented within existing HAI surveillance programs and has the potential to support infection-control efforts against this pathogen.

The Lifestyle Medicine Physician's Case to Self-Insured Employers: A Business Model for Physicians, a Bargain for Companies.

A majority of Americans have health care insurance through their employers, the cost for which is rising at an alarming rate. Unfortunately, higher expenditure on insurance has not translated into healthier employees. In this article, we present a value-based design plan for self-funded employers, administered by lifestyle medicine board-certified providers, which is purported to save money for employers and yet may demonstrate better health outcomes for employees by engaging them in evidence-based lifestyle interventions such as plant-based nutrition, regular physical activity and minimizing sedentary behaviors, sleeping well, tobacco cessation, and so on.

Interval analysis of blood filtration efficacy in novel device for autotransfusion in postpartum hemorrhage.

Postpartum haemorrhage (PPH), the leading cause of maternal mortality, is particularly problematic in low resource settings where access to safe blood supplies and definitive medical treatment is limited. We describe the continued development of an autotransfusion device designed to treat PPH by collection, filtration and infusion of maternal blood. Previous study has demonstrated that the device effectively moves blood through a filtration apparatus and removes up to 97% of aerobic bacteria but had poor anaerobic bacteria reduction. In this study, we investigate the filtration efficacy of the device using configurations comprised of three different leukocyte depletion filter designs: the Pall Leukoguard RS leukocyte reduction filter (PLRF), the Haemonetics BPF4™ (BPF4) leukocyte reduction filter, and the Haemonetics SCRC Leukotrap® (SCRC) filter. All configurations performed well with reductions ranging from 49 to 98%. Configurations containing 2 Haemonetics SCRC Leukotrap®filters (configuration 5 and 6) consistently reduced anaerobic bacteria by at least 73%. These results indicate that utilising a combination of SCRC and PLRF filters confers a high level of microbial filtration with improved removal of anaerobic organisms.

Silver-Coated Endotracheal Tubes Cleaned With a Mechanism for Secretion Removal.

BACKGROUND: Biofilm on the surface of endotracheal tubes (ETTs) is associated with ventilator-associated pneumonia. The use of silver-coated ETTs has been suggested to reduce the occurrence of ventilator-associated pneumonia by preventing biofilm formation. However, mucus accumulation can reduce the antibacterial activity of silver-coated ETTs by isolating bacterial colonies from the silver surface. We hypothesized that, in mechanically ventilated subjects, periodic removal of secretions through the use of a cleaning device would enhance the antimicrobial properties of silver-coated ETTs and thus reduce bacterial colonization. METHODS: Subjects were randomized to either standard suctioning (blind tracheal suctioning, control group) or blind tracheal suctioning plus cleaning maneuver every 8 h (treatment group). Tracheal aspirates were collected immediately before extubation for microbiological culture. After extubation, ETTs were collected for both cultural and non-cultural microbiological analysis and biofilm isolation. RESULTS: 39 subjects expected to be ventilated for > 48 h were enrolled; 36 ETTs (18 control, 18 treatment) and 29 tracheal samples (15 control, 14 treatment) were collected. Among the ETTs positive for bacterial colonization (15 vs 9, P = .18), cleaning maneuvers did not reduce microbial load, shown as the decimal logarithm of colony-forming units (CFU) per mL (1.6 ± 1.2 vs 0.9 ± 1.2 logCFU/mL, P = .15). There was a trend toward decreased biofilm deposition (439.5 ± 29.0 vs 288.9 ± 157.7 mg, P = .09) in the treated ETTs. No significant differences were observed in the number of positive tracheal aspirates (13 vs 10, P = .39) or in the microbial load (4.8 ± 4.0 vs 4.2 ± 3.8 logCFU/mL, P = .70) of tracheal secretions. Finally, no differences in the microbial load of Gram-positive organisms, Gram-negative organisms, or yeasts were found between the ETTs and tracheal aspirates of the 2 groups. CONCLUSIONS: In 39 critically-ill subjects intubated with silver-coated ETTs, periodic cleaning maneuvers did not decrease bacterial colonization of the ETTs and did not lower respiratory tract colonization compared to the standard suctioning. (Clinicaltrials.gov registration NCT02120001.).

Gnotobiotic and Conventional Mouse Systems to Support Microbiota Based Studies.

Animal models are essential to dissect host-microbiota interactions that impact health and the development of disease. In addition to providing pre-clinical models for the development of novel therapeutics and diagnostic biomarkers, mouse systems actively support microbiome studies by defining microbial contributions to normal development and homeostasis, and as well as their role in promoting diseases such as inflammatory auto-immune diseases, diabetes, metabolic syndromes, and susceptibilities to infectious agents. Mice provide a genetically tenable host that can be reared under gnotobiotic (germfree) conditions, allowing colonization studies with human or mouse-origin defined or complex microbial communities to define specific in vivo effects. The protocols and background information detail key aspects to consider in designing host-microbiome experiments with mouse models, and to develop robust systems that leverage gnotobiotic mice, microbial consortia, and specific environmental perturbations to identify causal effects in vivo.

Acetobacter indonesiensis Pneumonia after Lung Transplantation.

We report a case of Acetobacter indonesiensis pneumonia in a 51-year-old woman after bilateral lung transplantation. We found 2 other A. indonesiensis pneumonia cases reported in the literature. All 3 cases involved complex patients exposed to broad-spectrum antimicrobial drugs, suggesting that this pathogen may be opportunistic and highly drug-resistant.

Integrin-targeted cancer immunotherapy elicits protective adaptive immune responses.

Certain RGD-binding integrins are required for cell adhesion, migration, and proliferation and are overexpressed in most tumors, making them attractive therapeutic targets. However, multiple integrin antagonist drug candidates have failed to show efficacy in cancer clinical trials. In this work, we instead exploit these integrins as a target for antibody Fc effector functions in the context of cancer immunotherapy. By combining administration of an engineered mouse serum albumin/IL-2 fusion with an Fc fusion to an integrin-binding peptide (2.5F-Fc), significant survival improvements are achieved in three syngeneic mouse tumor models, including complete responses with protective immunity. Functional integrin antagonism does not contribute significantly to efficacy; rather, this therapy recruits both an innate and adaptive immune response, as deficiencies in either arm result in reduced tumor control. Administration of this integrin-targeted immunotherapy together with an anti-PD-1 antibody further improves responses and predominantly results in cures. Overall, this well-tolerated therapy achieves tumor specificity by redirecting inflammation to a functional target fundamental to tumorigenic processes but expressed at significantly lower levels in healthy tissues, and it shows promise for translation.

Multi-institute analysis of carbapenem resistance reveals remarkable diversity, unexplained mechanisms, and limited clonal outbreaks.

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by blaKPC2, blaKPC3, and blaKPC4, which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.

Episodic memory of odors stratifies Alzheimer biomarkers in normal elderly.

OBJECTIVE: The objective of this study was to relate a novel test of identifying and recalling odor percepts to biomarkers of Alzheimer's disease (AD) in well-characterized elderly individuals, ranging from cognitively normal to demented. METHODS: One hundred eighty-three participants (cognitively normal: n = 70; subjective cognitive concerns: n = 74; mild cognitive impairment [MCI]: n = 29, AD dementia: n = 10) were administered novel olfactory tests: the Odor Percept IDentification (OPID) and the Percepts of Odor Episodic Memory (POEM) tests. Univariate cross-sectional analyses of performance across diagnoses; logistic regression modeling, including covariates of age, sex, education, APOE genotype, and neuropsychological test scores; and linear mixed modeling of longitudinal cognitive scores were performed. Amyloid deposition and MRI volumetrics were analyzed in a subset of participants. RESULTS: Accuracy of identification and episodic memory of odor percepts differed significantly across diagnosis and age, with progressively worse performance across degrees of impairment. Among the participants who were cognitively normal or had subjective cognitive concerns, poorer than expected performance on the POEM test (based on the same individual's performance on the OPID and odor discrimination tests) was associated with higher frequencies of the APOE ε4 allele, thinner entorhinal cortices, and worse longitudinal trajectory of Logical Memory scores. INTERPRETATION: Selective impairment of episodic memory of odor percepts, relative to identification and discrimination of odor percepts revealed by this novel POEM battery, is associated with biomarkers of AD in a well-characterized pre-MCI population. These affordable, noninvasive olfactory tests offer potential to identify clinically normal individuals who have greater likelihood of future cognitive decline. Ann Neurol 2016;80:846-857.

MDSINE: Microbial Dynamical Systems INference Engine for microbiome time-series analyses.

Predicting dynamics of host-microbial ecosystems is crucial for the rational design of bacteriotherapies. We present MDSINE, a suite of algorithms for inferring dynamical systems models from microbiome time-series data and predicting temporal behaviors. Using simulated data, we demonstrate that MDSINE significantly outperforms the existing inference method. We then show MDSINE's utility on two new gnotobiotic mice datasets, investigating infection with Clostridium difficile and an immune-modulatory probiotic. Using these datasets, we demonstrate new capabilities, including accurate forecasting of microbial dynamics, prediction of stable sub-communities that inhibit pathogen growth, and identification of bacteria most crucial to community integrity in response to perturbations.

The Human Microbiome during Bacterial Vaginosis.

Bacterial vaginosis (BV) is the most commonly reported microbiological syndrome among women of childbearing age. BV is characterized by a shift in the vaginal flora from the dominant Lactobacillus to a polymicrobial flora. BV has been associated with a wide array of health issues, including preterm births, pelvic inflammatory disease, increased susceptibility to HIV infection, and other chronic health problems. A number of potential microbial pathogens, singly and in combinations, have been implicated in the disease process. The list of possible agents continues to expand and includes members of a number of genera, including Gardnerella, Atopobium, Prevotella, Peptostreptococcus, Mobiluncus, Sneathia, Leptotrichia, Mycoplasma, and BV-associated bacterium 1 (BVAB1) to BVAB3. Efforts to characterize BV using epidemiological, microscopic, microbiological culture, and sequenced-based methods have all failed to reveal an etiology that can be consistently documented in all women with BV. A careful analysis of the available data suggests that what we term BV is, in fact, a set of common clinical signs and symptoms that can be provoked by a plethora of bacterial species with proinflammatory characteristics, coupled to an immune response driven by variability in host immune function.